Multi-specific antigen-binding molecule having alternative function to function of blood coagulation factor viii

ABSTRACT

Various bispecific antibodies that specifically bind to both blood coagulation factor IX/activated blood coagulation factor IX and blood coagulation factor X and functionally substitute for the cofactor function of blood coagulation factor VIII, that is, the function to promote activation of blood coagulation factor X by activated blood coagulation factor IX, were produced. From these antibodies, multispecific antigen-binding molecules having a high activity of functionally substituting for blood coagulation factor VIII were successfully discovered.

TECHNICAL FIELD

The present invention relates to multispecific antigen-binding moleculesthat functionally substitute for blood coagulation factor VIII, acofactor that enhances enzymatic reactions, and pharmaceuticalcompositions comprising such a molecule as an active ingredient.

BACKGROUND ART

Hemophilia A is a bleeding abnormality caused by a hereditary decreaseor deficiency of blood coagulation factor VIII (F.VIII) function.Hemophilia A patients are generally administered with an F.VIIIformulation for the bleeding (on-demand administration). In recentyears, F.VIII formulations are also administered prophylactically toprevent bleeding events (preventive administration; Non-patent Documents1 and 2). The half-life of F.VIII formulations in blood is approximately12 to 16 hours. Therefore, for continuous prevention, F.VIIIformulations are administered to patients three times a week (Non-patentDocuments 3 and 4). In on-demand administrations, F.VIII formulationsare also additionally administered when necessary at regular intervalsto prevent rebleeding. In addition, the administration of F.VIIIformulations is done intravenously. Therefore, there has been a strongneed for pharmaceutical agents with a lesser burden than F.VIIIformulations.

Occasionally, anti-F.VIII antibodies (inhibitors) develop in hemophiliapatients. Such inhibitors cancel the effects of the F.VIII formulations.For bleeding in patients who have developed inhibitors (inhibitorpatients), bypass formulations are administered. Their action mechanismsare not dependent on F.VIII function, that is, the function ofcatalyzing the activation of blood coagulation factor X (F.X) byactivated blood coagulation factor IX (F.IXa). Therefore, in some cases,bypass formulations cannot sufficiently stop the bleeding. Accordingly,there has been a strong need for pharmaceutical agents that are notaffected by the presence of inhibitors and which can functionallysubstitute for F.VIII.

Recently, as a means for solving the problem, antibodies thatfunctionally substitute for F.VIII and their use were disclosed (PatentDocuments 1, 2, and 3). The antibodies may be effective for acquiredhemophilia in which anti-F.VIII autoantibodies are present and for vonWillebrand disease caused by an abnormality or deficiency of function ofvon Willebrand factor (vWF), but the activity of functionallysubstituting for F.VIII was not always sufficient. Therefore, aspharmaceutical agents exhibiting a high hemostatic effect, antibodieswith a higher activity of functionally substituting for F.VIII than theabove-mentioned antibodies were desired.

PRIOR ART DOCUMENTS Patent Document

-   [Patent Document 1] WO 2005/035754-   [Patent Document 2] WO 2005/035756-   [Patent Document 3] WO 2006/109592

Non-Patent Document

-   [Non-patent Document 1] Blood 58, 1-13 (1981)-   [Non-patent Document 2] Nature 312, 330-337 (1984)-   [Non-patent Document 3] Nature 312, 337-342 (1984)-   [Non-patent Document 4] Biochim. Biophys. Acta 871, 268-278 (1986)

SUMMARY OF THE INVENTION Problems to be Solved by the Invention

An objective of the present invention is to provide multispecificantigen-binding molecules that functionally substitute for F.VIII, acofactor that enhances enzymatic reactions.

Means for Solving the Problems

As a result of dedicated research, the present inventors succeeded indiscovering bispecific antibodies having a better F.Xageneration-promoting activity than known antibodies from among variousbispecific antibodies that specifically bind to both F.IX/F.IXa and F.X,and substitute for the cofactor function of F.VIII, that is, thefunction to promote F.X activation by F.IXa (F.Xa generation-promotingfunction).

Furthermore, the present inventors succeeded in finding the positions inthe amino acid sequences of bispecific antibodies having the activity offunctionally substituting for F.VIII that are important for improvingthe F.Xa generation-promoting activity of these antibodies, and thusthey successfully obtained bispecific antibodies in which the activityof functionally substituting for F.VIII is further increased byreplacing these amino acids. They also succeeded in obtaining bispecificantibodies which not only have a high activity of functionallysubstituting for F.VIII, but also have a low F.Xase inhibitory action.Satisfying both of these properties is very difficult.

Specifically, the present invention relates to multispecificantigen-binding molecules that functionally substitute for F.VIII, acofactor that enhances enzymatic reactions, and pharmaceuticalcompositions comprising such a molecule as an active ingredient, andspecifically relates to the following:

[1] a multispecific antigen-binding molecule that functionallysubstitutes for blood coagulation factor VIII, which comprises a firstantigen-binding site that recognizes blood coagulation factor IX and/oractivated blood coagulation factor IX and a second antigen-binding sitethat recognizes blood coagulation factor X, wherein the functionalsubstitution for blood coagulation factor VIII results from an activatedblood coagulation factor X (F.Xa) generation-promoting activity higherthan the activity of a bispecific antibody (hA69-KQ/hB26-PF/hAL-AQ)which comprises an H chain comprising SEQ ID NOs: 165 and 166, and acommonly shared L chain comprising SEQ ID NO: 167;[2] the multispecific antigen-binding molecule of [1], which comprises afirst polypeptide comprising a first antigen-binding site thatrecognizes blood coagulation factor IX and/or activated bloodcoagulation factor IX and a third polypeptide comprising a thirdantigen-binding site that recognizes blood coagulation factor IX and/oractivated blood coagulation factor IX, as well as a second polypeptidecomprising a second antigen-binding site that recognizes bloodcoagulation factor X and a fourth polypeptide comprising a fourthantigen-binding site that recognizes blood coagulation factor X;[3] the multispecific antigen-binding molecule of [2], wherein the firstpolypeptide and the third polypeptide each comprises an antigen-bindingsite of an H chain or L chain of an antibody against blood coagulationfactor IX or activated blood coagulation factor IX, respectively; andthe second polypeptide and the fourth polypeptide each comprises anantigen-binding site of an H chain or L chain of an antibody againstblood coagulation factor X, respectively;[4] the multispecific antigen-binding molecule of [3], wherein theantigen-binding site of the first polypeptide comprises anantigen-binding site which comprises H chain CDRs consisting of any oneof the amino acid sequences selected from the following (a1) to (a11),or an antigen-binding site functionally equivalent thereto, and theantigen-binding site of the second polypeptide comprises anantigen-binding site which comprises H chain CDRs consisting of any oneof the amino acid sequences selected from the following (b1) to (b11),or an antigen-binding site functionally equivalent thereto:

-   -   (a1) an antigen-binding site comprising an H chain CDR 1, 2, and        3 amino acid sequences of SEQ ID NOs: 75, 76, and 77 (H chain        CDRs of Q1), respectively;    -   (a2) an antigen-binding site comprising an H chain CDR 1, 2, and        3 amino acid sequences of SEQ ID NOs: 78, 79, and 80 (H chain        CDRs of Q31), respectively;    -   (a3) an antigen-binding site comprising an H chain CDR 1, 2, and        3 amino acid sequences of SEQ ID NOs: 81, 82, and 83 (H chain        CDRs of Q64), respectively;    -   (a4) an antigen-binding site comprising an H chain CDR 1, 2, and        3 amino acid sequences of SEQ ID NOs: 84, 85, and 86 (H chain        CDRs of Q85), respectively;    -   (a5) an antigen-binding site comprising the H chain CDR 1, 2,        and 3 amino acid sequences of SEQ ID NOs: 87, 88, and 89 (H        chain CDRs of Q153), respectively;    -   (a6) an antigen-binding site comprising an H chain CDR 1, 2, and        3 amino acid sequences of SEQ ID NOs: 90, 91, and 92 (H chain        CDRs of Q354), respectively;    -   (a7) an antigen-binding site comprising the H chain CDR 1, 2,        and 3 amino acid sequences of SEQ ID NOs: 93, 94, and 95 (H        chain CDRs of Q360), respectively;    -   (a8) an antigen-binding site comprising the of H chain CDR 1, 2,        and 3 amino acid sequences of SEQ ID NOs: 96, 97, and 98 (H        chain CDRs of Q405), respectively;    -   (a9) an antigen-binding site comprising an H chain CDR 1, 2, and        3 amino acid sequences of SEQ ID NOs: 99, 100, and 101 (H chain        CDRs of Q458), respectively;    -   (a10) an antigen-binding site comprising an H chain CDR 1, 2,        and 3 amino acid sequences of SEQ ID NOs: 102, 103, and 104 (H        chain CDRs of Q460), respectively;    -   (a11) an antigen-binding site comprising an H chain CDR 1, 2,        and 3 amino acid sequences of SEQ ID NOs: 105, 106, and 107 (H        chain CDRs of Q499), respectively;    -   (b1) an antigen-binding site comprising an H chain CDR 1, 2, and        3 amino acid sequences of SEQ ID NOs: 108, 109, and 110 (H chain        CDRs of J232), respectively;    -   (b2) an antigen-binding site comprising an H chain CDR 1, 2, and        3 amino acid sequences of SEQ ID NOs: 111, 112, and 113 (H chain        CDRs of J259), respectively;    -   (b3) an antigen-binding site comprising an H chain CDR 1, 2, and        3 amino acid sequences of SEQ ID NOs: 114, 115, and 116 (H chain        CDRs of J268), respectively;    -   (b4) an antigen-binding site comprising an H chain CDR 1, 2, and        3 amino acid sequences of SEQ ID NOs: 117, 118, and 119 (H chain        CDRs of J300), respectively;    -   (b5) an antigen-binding site comprising an H chain CDR 1, 2, and        3 amino acid sequences of SEQ ID NOs: 120, 121, and 122 (H chain        CDRs of J321), respectively;    -   (b6) an antigen-binding site comprising the H chain CDR 1, 2,        and 3 amino acid sequences of SEQ ID NOs: 123, 124, and 125 (H        chain CDRs of J326), respectively;    -   (b7) an antigen-binding site comprising an H chain CDR 1, 2, and        3 amino acid sequences of SEQ ID NOs: 126, 127, and 128 (H chain        CDRs of J327), respectively;    -   (b8) an antigen-binding site comprising an H chain CDR 1, 2, and        3 amino acid sequences of SEQ ID NOs: 129, 130, and 131 (H chain        CDRs of J339), respectively;    -   (b9) an antigen-binding site comprising an H chain CDR 1, 2, and        3 amino acid sequences of SEQ ID NOs: 132, 133, and 134 (H chain        CDRs of J344), respectively;    -   (b10) an antigen-binding site comprising an H chain CDR 1, 2,        and 3 amino acid sequences of SEQ ID NOs: 135, 136, and 137 (H        chain CDRs of J346), respectively; and    -   (b11) an antigen-binding site comprising an H chain CDR 1, 2,        and 3 amino acid sequences of SEQ ID NOs: 174, 175, and 176 (H        chain CDRs of J142), respectively;        [5] the multispecific antigen-binding molecule of [3], wherein        the antigen-binding site of the first polypeptide comprises an        antigen-binding site which comprises an H chain variable region        consisting of any one of the amino acid sequences selected from        the following (a1) to (a11), or an antigen-binding site        functionally equivalent thereto, and the antigen-binding site of        the second polypeptide comprises an antigen-binding site which        comprises an H chain variable region consisting of any one of        the amino acid sequences selected from the following (b1) to        (b11), or an antigen-binding site functionally equivalent        thereto:    -   (a1) an antigen-binding site comprising an H chain variable        region amino acid sequence of SEQ ID NO: 35 (H chain variable        region of Q1);    -   (a2) an antigen-binding site comprising an H chain variable        region amino acid sequence of SEQ ID NO: 36 (H chain variable        region of Q31);    -   (a3) an antigen-binding site comprising an H chain variable        region amino acid sequence of SEQ ID NO: 37 (H chain variable        region of Q1);    -   (a4) an antigen-binding site comprising an H chain variable        region amino acid sequence of SEQ ID NO: 38 (H chain variable        region of Q85);    -   (a5) an antigen-binding site comprising an H chain variable        region amino acid sequence of SEQ ID NO: 39 (H chain variable        region of Q153);    -   (a6) an antigen-binding site comprising an H chain variable        region amino acid sequence of SEQ ID NO: 40 (H chain variable        region of Q354);    -   (a7) an antigen-binding site comprising an H chain variable        region amino acid sequence of SEQ ID NO: 41 (H chain variable        region of Q360);    -   (a8) an antigen-binding site comprising an H chain variable        region amino acid sequence of SEQ ID NO: 42 (H chain variable        region of Q405);    -   (a9) an antigen-binding site comprising an H chain variable        region amino acid sequence of SEQ ID NO: 43 (H chain variable        region of Q458);    -   (a10) an antigen-binding site comprising an H chain variable        region amino acid sequence of SEQ ID NO: 44 (H chain variable        region of Q460);    -   (a11) an antigen-binding site comprising an H chain variable        region amino acid sequence of SEQ ID NO: 45 (H chain variable        region of Q499);    -   (b1) an antigen-binding site comprising an H chain variable        region amino acid sequence of SEQ ID NO: 46 (H chain variable        region of J232);    -   (b2) an antigen-binding site comprising an H chain variable        region amino acid sequence of SEQ ID NO: 47 (H chain variable        region of J259);    -   (b3) an antigen-binding site comprising an H chain variable        region amino acid sequence of SEQ ID NO: 48 (H chain variable        region of J268);    -   (b4) an antigen-binding site comprising an H chain variable        region amino acid sequence of SEQ ID NO: 49 (H chain variable        region of J300);    -   (b5) an antigen-binding site comprising an H chain variable        region amino acid sequence of SEQ ID NO: 50 (H chain variable        region of J321);    -   (b6) an antigen-binding site comprising an H chain variable        region amino acid sequence of SEQ ID NO: 51 (H chain variable        region of J326);    -   (b7) an antigen-binding site comprising an H chain variable        region amino acid sequence of SEQ ID NO: 52 (H chain variable        region of J327);    -   (b8) an antigen-binding site comprising an H chain variable        region amino acid sequence of SEQ ID NO: 53 (H chain variable        region of J339);    -   (b9) an antigen-binding site comprising an H chain variable        region amino acid sequence of SEQ ID NO: 54 (H chain variable        region of J344);    -   (b10) an antigen-binding site comprising an H chain variable        region amino acid sequence of SEQ ID NO: 55 (H chain variable        region of J346); and    -   (b11) an antigen-binding site comprising an H chain variable        region amino acid sequence of SEQ ID NO: 172 (H chain variable        region of J142);        [6] the multispecific antigen-binding molecule of [3], wherein        the antigen-binding sites included in the third polypeptide and        the fourth polypeptide comprise an antigen-binding site which        comprises L chain CDRs consisting of any one of the amino acid        sequences selected from the following (c1) to (c10), or an        antigen-binding site functionally equivalent thereto:    -   (c1) an antigen-binding site comprising an L chain CDR1, 2, and        3 amino acid sequences of SEQ ID NOs: 138, 139, and 140 (L chain        CDR of L2), respectively;    -   (c2) an antigen-binding site comprising an L chain CDR1, 2, and        3 amino acid sequences of SEQ ID NOs: 141, 142, and 143 (L chain        CDR of L45), respectively;    -   (c3) an antigen-binding site comprising an L chain CDR1, 2, and        3 amino acid sequences of SEQ ID NOs: 144, 145, and 146 (L chain        CDR of L248), respectively;    -   (c4) an antigen-binding site comprising an L chain CDR1, 2, and        3 amino acid sequences of SEQ ID NOs: 147, 148, and 149 (L chain        CDR of L324), respectively;    -   (c5) an antigen-binding site comprising an L chain CDR1, 2, and        3 amino acid sequences of SEQ ID NOs: 150, 151, and 152 (L chain        CDR of L334), respectively;    -   (c6) an antigen-binding site comprising an L chain CDR1, 2, and        3 amino acid sequences of SEQ ID NOs: 153, 154, and 155 (L chain        CDR of L377), respectively;    -   (c7) an antigen-binding site comprising an L chain CDR1, 2, and        3 amino acid sequences of SEQ ID NOs: 156, 157, and 158 (L chain        CDR of L404), respectively;    -   (c8) an antigen-binding site comprising an L chain CDR1, 2, and        3 amino acid sequences of SEQ ID NOs: 159, 160, and 161 (L chain        CDR of L406), respectively;    -   (c9) an antigen-binding site comprising an L chain CDR1, 2, and        3 amino acid sequences of SEQ ID NOs: 137, 138, and 139 (L chain        CDR of L408), respectively; and    -   (c10) an antigen-binding site comprising an L chain CDR1, 2, and        3 amino acid sequences of SEQ ID NOs: 177, 178, and 179 (L chain        CDR of L180), respectively;        [7] the multispecific antigen-binding molecule of [3], wherein        the antigen-binding sites included in the third polypeptide and        the fourth polypeptide comprise an antigen-binding site which        comprises an L chain variable region consisting of any one of        the amino acid sequences selected from the following (c1) to        (c10), or an antigen-binding site functionally equivalent        thereto:    -   (c1) an antigen-binding site comprising an L chain variable        region amino acid sequence of SEQ ID NO: 56 (L chain variable        region of L2);    -   (c2) an antigen-binding site comprising an L chain variable        region amino acid sequence of SEQ ID NO: 57 (L chain variable        region of L45);    -   (c3) an antigen-binding site comprising an L chain variable        region amino acid sequence of SEQ ID NO: 58 (L chain variable        region of L248);    -   (c4) an antigen-binding site comprising an L chain variable        region amino acid sequence of SEQ ID NO: 59 (L chain variable        region of L324);    -   (c5) an antigen-binding site comprising an L chain variable        region amino acid sequence of SEQ ID NO: 60 (L chain variable        region of L334);    -   (c6) an antigen-binding site comprising an L chain variable        region amino acid sequence of SEQ ID NO: 61 (L chain variable        region of L377);    -   (c7) an antigen-binding site comprising an L chain variable        region amino acid sequence of SEQ ID NO: 62 (L chain variable        region of L404);    -   (c8) an antigen-binding site comprising an L chain variable        region amino acid sequence of SEQ ID NO: 63 (L chain variable        region of L406);    -   (c9) an antigen-binding site comprising an L chain variable        region amino acid sequence of SEQ ID NO: 64 (L chain variable        region of L408); and    -   (c10) an antigen-binding site comprising an L chain variable        region amino acid sequence of SEQ ID NO: 173 (L chain variable        region of L180);        [8] the multispecific antigen-binding molecule of [3], wherein        the first and second polypeptides further comprise an antibody H        chain constant region, and the third and fourth polypeptides        comprise an antibody L chain constant region;        [9] the multispecific antigen-binding molecule of [3], wherein        the first and second polypeptides comprise an antibody H chain        constant region, and the third and fourth polypeptides comprise        an antibody L chain constant region, and wherein the third        polypeptide and the fourth polypeptide are a commonly shared L        chain;        [10] the multispecific antigen-binding molecule of [8] or [9],        wherein the first polypeptide comprises an antibody H chain        constant region consisting of any one of the amino acid        sequences selected from the group consisting of the following        (d1) to (d6) or the group consisting of the following (d7) to        (d9), and the second polypeptide comprises an antibody H chain        constant region consisting of any one of the amino acid        sequences selected from a group different from that of the        above-mentioned first polypeptide:    -   (d1) an H chain constant region of SEQ ID NO: 65 (G4k);    -   (d2) an H chain constant region of SEQ ID NO: 66 (z7);    -   (d3) an H chain constant region of SEQ ID NO: 67 (z55);    -   (d4) an H chain constant region of SEQ ID NO: 68 (z106);    -   (d5) an H chain constant region of SEQ ID NO: 69 (z118);    -   (d6) an H chain constant region of SEQ ID NO: 70 (z121);    -   (d7) an H chain constant region of SEQ ID NO: 71 (G4h);    -   (d8) an H chain constant region of SEQ ID NO: 72 (z107); and    -   (d9) an H chain constant region of SEQ ID NO: 73 (z119);        [11] the multispecific antigen-binding molecule of [8] or [9],        wherein the third and fourth polypeptides comprise the antibody        L chain constant region consisting of the following amino acid        sequence of:        (e) an L chain constant region of SEQ ID NO: 74 (k);        [12] the multispecific antigen-binding molecule of [8] or [9],        wherein the first polypeptide comprises any one antibody H chain        selected from the following (a1) to (a14), the second        polypeptide comprises any one antibody H chain selected from the        following (b1) to (b12), and the third polypeptide and the        fourth polypeptide comprise any one antibody L chain selected        from the following (c1) to (c10):    -   (a1) an antibody H chain consisting of the amino acid sequence        of SEQ ID NO: 1 (Q1-G4k);    -   (a2) an antibody H chain consisting of the amino acid sequence        of SEQ ID NO: 2 (Q31-z7);    -   (a3) an antibody H chain consisting of the amino acid sequence        of SEQ ID NO: 3 (Q64-z55);    -   (a4) an antibody H chain consisting of the amino acid sequence        of SEQ ID NO: 10 (Q64-z7);    -   (a5) an antibody H chain consisting of the amino acid sequence        of SEQ ID NO: 11 (Q85-G4k);    -   (a6) an antibody H chain consisting of the amino acid sequence        of SEQ ID NO: 12 (Q153-G4k);    -   (a7) an antibody H chain consisting of the amino acid sequence        of SEQ ID NO: 13 (Q354-z106);    -   (a8) an antibody H chain consisting of the amino acid sequence        of SEQ ID NO: 14 (Q360-G4k);    -   (a9) an antibody H chain consisting of the amino acid sequence        of SEQ ID NO: 15 (Q360-z118);    -   (a10) an antibody H chain consisting of the amino acid sequence        of SEQ ID NO: 16 (Q405-G4k);    -   (a11) an antibody H chain consisting of the amino acid sequence        of SEQ ID NO: 17 (Q458-z106);    -   (a12) an antibody H chain consisting of the amino acid sequence        of SEQ ID NO: 18 (Q460-z121);    -   (a13) an antibody H chain consisting of the amino acid sequence        of SEQ ID NO: 19 (Q499-z118);    -   (a14) an antibody H chain consisting of the amino acid sequence        of SEQ ID NO: 20 (Q499-z121);    -   (b1) an antibody H chain consisting of the amino acid sequence        of SEQ ID NO: 4 (J268-G4h);    -   (b2) an antibody H chain consisting of the amino acid sequence        of SEQ ID NO: 5 (J321-G4h);    -   (b3) an antibody H chain consisting of the amino acid sequence        of SEQ ID NO: 6 (J326-z107);    -   (b4) an antibody H chain consisting of the amino acid sequence        of SEQ ID NO: 7 (J344-z107);    -   (b5) an antibody H chain consisting of the amino acid sequence        of SEQ ID NO: 21 (J232-G4h);    -   (b6) an antibody H chain consisting of the amino acid sequence        of SEQ ID NO: 22 (J259-z107);    -   (b7) an antibody H chain consisting of the amino acid sequence        of SEQ ID NO: 23 (J300-z107);    -   (b8) an antibody H chain consisting of the amino acid sequence        of SEQ ID NO: 24 (J327-z107);    -   (b9) an antibody H chain consisting of the amino acid sequence        of SEQ ID NO: 25 (J327-z119);    -   (b10) an antibody H chain consisting of the amino acid sequence        of SEQ ID NO: 26 (J339-z119);    -   (b11) an antibody H chain consisting of the amino acid sequence        of SEQ ID NO: 27 (J346-z107);    -   (b12) an antibody H chain consisting of the amino acid sequence        of SEQ ID NO: 170 (J142-G4h);    -   (c1) an antibody L chain consisting of the amino acid sequence        of SEQ ID NO: 8 (L2-k);    -   (c2) an antibody L chain consisting of the amino acid sequence        of SEQ ID NO: 9 (L45-k);    -   (c3) an antibody L chain consisting of the amino acid sequence        of SEQ ID NO: 28 (L248-k);    -   (c4) an antibody L chain consisting of the amino acid sequence        of SEQ ID NO: 29 (L324-k);    -   (c5) an antibody L chain consisting of the amino acid sequence        of SEQ ID NO: 30 (L334-k);    -   (c6) an antibody L chain consisting of the amino acid sequence        of SEQ ID NO: 31 (L377-k);    -   (c7) an antibody L chain consisting of the amino acid sequence        of SEQ ID NO: 32 (L404-k);    -   (c8) an antibody L chain consisting of the amino acid sequence        of SEQ ID NO: 33 (L406-k);    -   (c9) an antibody L chain consisting of the amino acid sequence        of SEQ ID NO: 34 (L408-k); and    -   (c10) an antibody L chain consisting of the amino acid sequence        of SEQ ID NO: 171 (L180-k);        [13] the multispecific antigen-binding molecule of [1], wherein        the first polypeptide comprises an antigen-binding site which        binds to an epitope overlapping with an epitope that binds to an        antibody consisting of the antibody H chain of any one of (a1)        to (a14) and the antibody L chain of any one of (c1) to (c10) of        [12], and the second polypeptide comprises an antigen-binding        site which binds to an epitope overlapping with an epitope that        binds to an antibody consisting of the antibody H chain of any        one of (b1) to (b12) and the antibody L chain of any one of (c1)        to (c10) of [12];        [14] the multispecific antigen-binding molecule of [8] or [9],        wherein the first polypeptide comprises any one antibody H chain        selected from the following (e1) to (e3), the second polypeptide        comprises any one antibody H chain selected from the following        (f1) to (f3), and the third polypeptide and the fourth        polypeptide comprise any one antibody L chain selected from the        following (g1) to (g4):    -   (e1) an H chain of an antibody which binds to an epitope        overlapping with an epitope bound by an antibody consisting of        an antibody H chain of any one of (a1) to (a14) and an antibody        L chain of any one of (c1) to (c10), of [12];    -   (e2) an antibody H chain, wherein at least one amino acid        residue selected from the amino acid residues at positions 34,        35, 49, 61, 62, 96, 98, 100, 100b, and 102 by Kabat numbering in        any one antibody H chain selected from (e1) is substituted with        another amino acid;    -   (e3) an antibody H chain, wherein by Kabat numbering, the amino        acid residue at position 34 is isoleucine, the amino acid        residue at position 35 is asparagine, glutamine, or serine, the        amino acid residue at position 49 is serine, the amino acid        residue at position 61 is arginine, the amino acid residue at        position 62 is glutamic acid, the amino acid residue at position        96 is serine or threonine, the amino acid residue at position 98        is lysine or arginine, the amino acid residue at position 100 is        phenylalanine or tyrosine, the amino acid residue at position        100b is glycine, or the amino acid residue at position 102 is        tyrosine in any antibody H chain selected from (e1);    -   (f1) an H chain of an antibody which binds to an epitope        overlapping with an epitope bound by an antibody consisting of        an antibody H chain of any of (b1) to (b12) of [12] and an        antibody L chain of any of (c1) to (c10) of [12];    -   (f2) an antibody H chain, wherein at least one amino acid        residue selected from the amino acid residues at positions 35,        53, 73, 76, 96, 98, 100, and 100a by Kabat numbering in any        antibody H chain of (f1) is substituted with another amino acid;    -   (f3) an antibody H chain, wherein by Kabat numbering, the amino        acid residue at position 35 is aspartic acid, the amino acid        residue at position 53 is arginine, the amino acid residue at        position 73 is lysine, the amino acid residue at position 76 is        glycine, the amino acid residue at position 96 is lysine or        arginine, the amino acid residue at position 98 is tyrosine, the        amino acid residue at position 100 is tyrosine, or the amino        acid residue at position 100a is histidine in any one antibody H        chain selected from (f1);    -   (g1) an L chain of an antibody which binds to an epitope        overlapping with an epitope bound by an antibody which consists        of an antibody H chain of any one of (a1) to (a14) and an        antibody L chain of any one of (c1) to (c10), of [12];    -   (g2) an L chain of an antibody which binds to an epitope        overlapping with an epitope bound by an antibody which consists        of an antibody H chain of any one of (b1) to (b12) and an        antibody L chain of any one of (c1) to (c10), of [12];    -   (g3) an antibody L chain, wherein at least one amino acid        residue selected from the amino acid residues at positions 27,        30, 31, 32, 50, 52, 53, 54, 55, 92, 93, 94, and 95 by Kabat        numbering in the antibody L chain of either (g1) or (g2) is        substituted with another amino acid; and    -   (g4) an antibody L chain, wherein by Kabat numbering, the amino        acid residue at position 27 is lysine or arginine, the amino        acid residue at position 30 is glutamic acid, the amino acid        residue at position 31 is arginine, the amino acid residue at        position 32 is glutamine, the amino acid residue at position 50        is arginine or glutamine, the amino acid residue at position 52        is serine, the amino acid residue at position 53 is arginine,        the amino acid residue at position 54 is lysine, the amino acid        residue at position 55 is glutamic acid, the amino acid residue        at position 92 is serine, the amino acid residue at position 93        is serine, the amino acid residue at position 94 is proline, or        the amino acid residue at position 95 is proline in the antibody        L chain of either (g1) or (g2);        [15] the multispecific antigen-binding molecule of any one of        [1] to [14], wherein the multispecific antigen-binding molecule        is a multispecific antibody;        [16] a bispecific antibody of any one of the following (a) to        (u):    -   (a) a bispecific antibody (Q1-G4k/J268-G4h/L45-k), wherein the        first polypeptide is an H chain consisting of the amino acid        sequence of SEQ ID NO: 1, the second polypeptide is an H chain        consisting of the amino acid sequence of SEQ ID NO: 4, and the        third polypeptide and the fourth polypeptide are a commonly        shared L chain of SEQ ID NO: 9;    -   (b) a bispecific antibody (Q1-G4k/J321-G4h/L45-k), wherein the        first polypeptide is an H chain consisting of the amino acid        sequence of SEQ ID NO: 1, the second polypeptide is an H chain        consisting of the amino acid sequence of SEQ ID NO: 5, and the        third polypeptide and the fourth polypeptide are a commonly        shared L chain of SEQ ID NO: 9;    -   (c) a bispecific antibody (Q31-z7/J326-z107/L2-k), wherein the        first polypeptide is an H chain consisting of the amino acid        sequence of SEQ ID NO: 2, the second polypeptide is an H chain        consisting of the amino acid sequence of SEQ ID NO: 6, and the        third polypeptide and the fourth polypeptide are a commonly        shared L chain of SEQ ID NO: 8;    -   (d) a bispecific antibody (Q64-z55/J344-z107/L45-k), wherein the        first polypeptide is an H chain consisting of the amino acid        sequence of SEQ ID NO: 3, the second polypeptide is an H chain        consisting of the amino acid sequence of SEQ ID NO: 7, and the        third polypeptide and the fourth polypeptide are a commonly        shared L chain of SEQ ID NO: 9;    -   (e) a bispecific antibody (Q64-z7/J326-z107/L334-k), wherein the        first polypeptide is an H chain consisting of the amino acid        sequence of SEQ ID NO: 10, the second polypeptide is an H chain        consisting of the amino acid sequence of SEQ ID NO: 6, and the        third polypeptide and the fourth polypeptide are a commonly        shared L chain of SEQ ID NO: 30;    -   (f) a bispecific antibody (Q64-z7/J344-z107/L406-k), wherein the        first polypeptide is an H chain consisting of the amino acid        sequence of SEQ ID NO: 10, the second polypeptide is an H chain        consisting of the amino acid sequence of SEQ ID NO: 7, and the        third polypeptide and the fourth polypeptide are a commonly        shared L chain of SEQ ID NO: 33;    -   (g) a bispecific antibody (Q85-G4k/J268-G4h/L406-k), wherein the        first polypeptide is an H chain consisting of the amino acid        sequence of SEQ ID NO: 11, the second polypeptide is an H chain        consisting of the amino acid sequence of SEQ ID NO: 4, and the        third polypeptide and the fourth polypeptide are a commonly        shared L chain of SEQ ID NO: 33;    -   (h) a bispecific antibody (Q85-G4k/J321-G4h/L334-k), wherein the        first polypeptide is an H chain consisting of the amino acid        sequence of SEQ ID NO: 11, the second polypeptide is an H chain        consisting of the amino acid sequence of SEQ ID NO: 5, and the        third polypeptide and the fourth polypeptide are a commonly        shared L chain of SEQ ID NO: 30;    -   (i) a bispecific antibody (Q153-G4k/J232-G4h/L406-k), wherein        the first polypeptide is an H chain consisting of the amino acid        sequence of SEQ ID NO: 12, the second polypeptide is an H chain        consisting of the amino acid sequence of SEQ ID NO: 21, and the        third polypeptide and the fourth polypeptide are a commonly        shared L chain of SEQ ID NO: 33;    -   (j) a bispecific antibody (Q354-z106/J259-z107/L324-k), wherein        the first polypeptide is an H chain consisting of the amino acid        sequence of SEQ ID NO: 13, the second polypeptide is an H chain        consisting of the amino acid sequence of SEQ ID NO: 22, and the        third polypeptide and the fourth polypeptide are a commonly        shared L chain of SEQ ID NO: 29;    -   (k) a bispecific antibody (Q360-G4k/J232-G4h/L406-k), wherein        the first polypeptide is an H chain consisting of the amino acid        sequence of SEQ ID NO: 14, the second polypeptide is an H chain        consisting of the amino acid sequence of SEQ ID NO: 21, and the        third polypeptide and the fourth polypeptide are a commonly        shared L chain of SEQ ID NO: 33;    -   (l) a bispecific antibody (Q360-z118/J300-z107/L334-k), wherein        the first polypeptide is an H chain consisting of the amino acid        sequence of SEQ ID NO: 15, the second polypeptide is an H chain        consisting of the amino acid sequence of SEQ ID NO: 23, and the        third polypeptide and the fourth polypeptide are a commonly        shared L chain of SEQ ID NO: 30;    -   (m) a bispecific antibody (Q405-G4k/J232-G4h/L248-k), wherein        the first polypeptide is an H chain consisting of the amino acid        sequence of SEQ ID NO: 16, the second polypeptide is an H chain        consisting of the amino acid sequence of SEQ ID NO: 21, and the        third polypeptide and the fourth polypeptide are a commonly        shared L chain of SEQ ID NO: 28;    -   (n) a bispecific antibody (Q458-z106/J346-z107/L408-k), wherein        the first polypeptide is an H chain consisting of the amino acid        sequence of SEQ ID NO: 17, the second polypeptide is an H chain        consisting of the amino acid sequence of SEQ ID NO: 27, and the        third polypeptide and the fourth polypeptide are a commonly        shared L chain of SEQ ID NO: 34;    -   (o) a bispecific antibody (Q460-z121/J327-z119/L334-k), wherein        the first polypeptide is an H chain consisting of the amino acid        sequence of SEQ ID NO: 18, the second polypeptide is an H chain        consisting of the amino acid sequence of SEQ ID NO: 25, and the        third polypeptide and the fourth polypeptide are a commonly        shared L chain of SEQ ID NO: 30;    -   (p) a bispecific antibody (Q499-z118/J327-z107/L334-k), wherein        the first polypeptide is an H chain consisting of the amino acid        sequence of SEQ ID NO: 19, the second polypeptide is an H chain        consisting of the amino acid sequence of SEQ ID NO: 24, and the        third polypeptide and the fourth polypeptide are a commonly        shared L chain of SEQ ID NO: 30;    -   (q) a bispecific antibody (Q499-z118/J327-z107/L377-k), wherein        the first polypeptide is an H chain consisting of the amino acid        sequence of SEQ ID NO: 19, the second polypeptide is an H chain        consisting of the amino acid sequence of SEQ ID NO: 24, and the        third polypeptide and the fourth polypeptide are a commonly        shared L chain of SEQ ID NO: 31;    -   (r) a bispecific antibody (Q499-z118/J346-z107/L248-k), wherein        the first polypeptide is an H chain consisting of the amino acid        sequence of SEQ ID NO: 19, the second polypeptide is an H chain        consisting of the amino acid sequence of SEQ ID NO: 27, and the        third polypeptide and the fourth polypeptide are a commonly        shared L chain of SEQ ID NO: 28;    -   (s) a bispecific antibody (Q499-z121/J327-z119/L404-k), wherein        the first polypeptide is an H chain consisting of the amino acid        sequence of SEQ ID NO: 20, the second polypeptide is an H chain        consisting of the amino acid sequence of SEQ ID NO: 25, and the        third polypeptide and the fourth polypeptide are a commonly        shared L chain of SEQ ID NO: 32;    -   (t) a bispecific antibody (Q499-z121/J339-z119/L377-k), wherein        the first polypeptide is an H chain consisting of the amino acid        sequence of SEQ ID NO: 20, the second polypeptide is an H chain        consisting of the amino acid sequence of SEQ ID NO: 26, and the        third polypeptide and the fourth polypeptide are a commonly        shared L chain of SEQ ID NO: 31; and    -   (u) a bispecific antibody (Q153-G4k/J142-G4h/L180-k), wherein        the first polypeptide is an H chain consisting of the amino acid        sequence of SEQ ID NO: 12, the second polypeptide is an H chain        consisting of the amino acid sequence of SEQ ID NO: 170, and the        third polypeptide and the fourth polypeptide are a commonly        shared L chain of SEQ ID NO: 171;        [17] a nucleic acid encoding the multispecific antigen-binding        molecule of any one of [1] to [15] or the bispecific antibody of        [16];        [18] a vector inserted with the nucleic acid of [17];        [19] a cell comprising the nucleic acid of [17] or the vector of        [18];        [20] a method for producing the multispecific antigen-binding        molecule of any one of [1] to [15] or the bispecific antibody of        [16] by culturing the cell of [19];        [21] a pharmaceutical composition comprising the multispecific        antigen-binding molecule of any one of [1] to [15] or the        bispecific antibody of [16], and a pharmaceutically acceptable        carrier;        [22] the composition of [21], which is a pharmaceutical        composition used for prevention and/or treatment of bleeding, a        disease accompanying bleeding, or a disease caused by bleeding;        [23] the composition of [22], wherein the bleeding, the disease        accompanying bleeding, or the disease caused by bleeding is a        disease that develops and/or progresses due to a decrease or        deficiency in the activity of blood coagulation factor VIII        and/or activated blood coagulation factor VIII;        [24] the composition of [23], wherein the disease that develops        and/or progresses due to a decrease or deficiency in the        activity of blood coagulation factor VIII and/or activated blood        coagulation factor VIII is hemophilia A;        [25] the composition of [23], wherein the disease that develops        and/or progresses due to a decrease or deficiency in the        activity of blood coagulation factor VIII and/or activated blood        coagulation factor VIII is a disease showing emergence of an        inhibitor against blood coagulation factor VIII and/or activated        blood coagulation factor VIII;        [26] the composition of [23], wherein the disease that develops        and/or progresses due to a decrease or deficiency in the        activity of blood coagulation factor VIII and/or activated blood        coagulation factor VIII is acquired hemophilia;        [27] the composition of [23], wherein the disease that develops        and/or progresses due to a decrease in the activity of blood        coagulation factor VIII and/or activated blood coagulation        factor VIII is von Willebrand disease;        [28] a method for preventing and/or treating bleeding, a disease        accompanying bleeding, or a disease caused by bleeding, which        comprises the step of administering the multispecific        antigen-binding molecule of any one of [1] to [15] or the        bispecific antibody of [16], or the composition of any one of        [21] to [27]; and        [29] a kit for use in the prevention and/or treatment method of        [28], which comprises at least the multispecific antigen-binding        molecule of any one of [1] to [15] or the bispecific antibody of        [16], or the composition of any one of [21] to [27].

Furthermore, the present invention relates to:

[30] use of the multispecific antigen-binding molecule of any one of [1]to [15], the bispecific antibody of [16], or the composition of any oneof [21] to [27] in the manufacture of an agent for preventing and/ortreating bleeding, a disease accompanying bleeding, or a disease causedby bleeding; and[31] the multispecific antigen-binding molecule of any one of [1] to[15], the bispecific antibody of [16], or the composition of any one of[21] to [27] for preventing and/or treating bleeding, a diseaseaccompanying bleeding, or a disease caused by bleeding.

The present invention also relates to bispecific antibodies thatfunctionally substitute for F.VIII, a cofactor that enhances enzymaticreactions, and pharmaceutical compositions comprising the antibody as anactive ingredient, and more specifically relates to:

[32] a bispecific antibody that functionally substitutes for bloodcoagulation factor VIII, which comprises a first antigen-binding sitethat recognizes blood coagulation factor IX and/or activated bloodcoagulation factor IX and a second antigen-binding site that recognizesblood coagulation factor X, wherein the bispecific antibody is any ofthe following (a) to (u):

-   -   (a) a bispecific antibody (Q1-G4k/J268-G4h/L45-k), wherein the        first polypeptide is an H chain consisting of the amino acid        sequence of SEQ ID NO: 1, the second polypeptide is an H chain        consisting of the amino acid sequence of SEQ ID NO: 4, and the        third polypeptide and the fourth polypeptide are a commonly        shared L chain of SEQ ID NO: 9;    -   (b) a bispecific antibody (Q1-G4k/J321-G4h/L45-k), wherein the        first polypeptide is an H chain consisting of the amino acid        sequence of SEQ ID NO: 1, the second polypeptide is an H chain        consisting of the amino acid sequence of SEQ ID NO: 5, and the        third polypeptide and the fourth polypeptide are a commonly        shared L chain of SEQ ID NO: 9;    -   (c) a bispecific antibody (Q31-z7/J326-z107/L2-k), wherein the        first polypeptide is an H chain consisting of the amino acid        sequence of SEQ ID NO: 2, the second polypeptide is an H chain        consisting of the amino acid sequence of SEQ ID NO: 6, and the        third polypeptide and the fourth polypeptide are a commonly        shared L chain of SEQ ID NO: 8;    -   (d) a bispecific antibody (Q64-z55/J344-z107/L45-k), wherein the        first polypeptide is an H chain consisting of the amino acid        sequence of SEQ ID NO: 3, the second polypeptide is an H chain        consisting of the amino acid sequence of SEQ ID NO: 7, and the        third polypeptide and the fourth polypeptide are a commonly        shared L chain of SEQ ID NO: 9;    -   (e) a bispecific antibody (Q64-z7/J326-z107/L334-k), wherein the        first polypeptide is an H chain consisting of the amino acid        sequence of SEQ ID NO: 10, the second polypeptide is an H chain        consisting of the amino acid sequence of SEQ ID NO: 6, and the        third polypeptide and the fourth polypeptide are a commonly        shared L chain of SEQ ID NO: 30;    -   (f) a bispecific antibody (Q64-z7/J344-z107/L406-k), wherein the        first polypeptide is an H chain consisting of the amino acid        sequence of SEQ ID NO: 10, the second polypeptide is an H chain        consisting of the amino acid sequence of SEQ ID NO: 7, and the        third polypeptide and the fourth polypeptideare a commonly        shared L chain of SEQ ID NO: 33;    -   (g) a bispecific antibody (Q85-G4k/J268-G4h/L406-k), wherein the        first polypeptide is an H chain consisting of the amino acid        sequence of SEQ ID NO: 11, the second polypeptide is an H chain        consisting of the amino acid sequence of SEQ ID NO: 4, and the        third polypeptide and the fourth polypeptide are a commonly        shared L chain of SEQ ID NO: 33;    -   (h) a bispecific antibody (Q85-G4k/J321-G4h/L334-k), wherein the        first polypeptide is an H chain consisting of the amino acid        sequence of SEQ ID NO: 11, the second polypeptide is an H chain        consisting of the amino acid sequence of SEQ ID NO: 5, and the        third polypeptide and the fourth polypeptide are a commonly        shared L chain of SEQ ID NO: 30;    -   (i) a bispecific antibody (Q153-G4k/J232-G4h/L406-k), wherein        the first polypeptide is an H chain consisting of the amino acid        sequence of SEQ ID NO: 12, the second polypeptide is an H chain        consisting of the amino acid sequence of SEQ ID NO: 21, and the        third polypeptide and the fourth polypeptide are a commonly        shared L chain of SEQ ID NO: 33;    -   (j) a bispecific antibody (Q354-z106/J259-z107/L324-k), wherein        the first polypeptide is an H chain consisting of the amino acid        sequence of SEQ ID NO: 13, the second polypeptide is an H chain        consisting of the amino acid sequence of SEQ ID NO: 22, and the        third polypeptide and the fourth polypeptide are a commonly        shared L chain of SEQ ID NO: 29;    -   (k) a bispecific antibody (Q360-G4k/J232-G4h/L406-k), wherein        the first polypeptide is an H chain consisting of the amino acid        sequence of SEQ ID NO: 14, the second polypeptide is an H chain        consisting of the amino acid sequence of SEQ ID NO: 21, and the        third polypeptide and the fourth polypeptide are a commonly        shared L chain of SEQ ID NO: 33;    -   (l) a bispecific antibody (Q360-z118/J300-z107/L334-k), wherein        the first polypeptide is an H chain consisting of the amino acid        sequence of SEQ ID NO: 15, the second polypeptide is an H chain        consisting of the amino acid sequence of SEQ ID NO: 23, and the        third polypeptide and the fourth polypeptide are a commonly        shared L chain of SEQ ID NO: 30;    -   (m) a bispecific antibody (Q405-G4k/J232-G4h/L248-k), wherein        the first polypeptide is an H chain consisting of the amino acid        sequence of SEQ ID NO: 16, the second polypeptide is an H chain        consisting of the amino acid sequence of SEQ ID NO: 21, and the        third polypeptide and the fourth polypeptide are a commonly        shared L chain of SEQ ID NO: 28;    -   (n) a bispecific antibody (Q458-z106/J346-z107/L408-k), wherein        the first polypeptide is an H chain consisting of the amino acid        sequence of SEQ ID NO: 17, the second polypeptide is an H chain        consisting of the amino acid sequence of SEQ ID NO: 27, and the        third polypeptide and the fourth polypeptide are a commonly        shared L chain of SEQ ID NO: 34;    -   (o) a bispecific antibody (Q460-z121/J327-z119/L334-k), wherein        the first polypeptide is an H chain consisting of the amino acid        sequence of SEQ ID NO: 18, the second polypeptide is an H chain        consisting of the amino acid sequence of SEQ ID NO: 25, and the        third polypeptide and the fourth polypeptide are a commonly        shared L chain of SEQ ID NO: 30;    -   (p) a bispecific antibody (Q499-z118/J327-z107/L334-k), wherein        the first polypeptide is an H chain consisting of the amino acid        sequence of SEQ ID NO: 19, the second polypeptide is an H chain        consisting of the amino acid sequence of SEQ ID NO: 24, and the        third polypeptide and the fourth polypeptide are a commonly        shared L chain of SEQ ID NO: 30;    -   (q) a bispecific antibody (Q499-z118/J327-z107/L377-k), wherein        the first polypeptide is an H chain consisting of the amino acid        sequence of SEQ ID NO: 19, the second polypeptide is an H chain        consisting of the amino acid sequence of SEQ ID NO: 24, and the        third polypeptide and the fourth polypeptide are a commonly        shared L chain of SEQ ID NO: 31;    -   (r) a bispecific antibody (Q499-z118/J346-z107/L248-k), wherein        the first polypeptide is an H chain consisting of the amino acid        sequence of SEQ ID NO: 19, the second polypeptide is an H chain        consisting of the amino acid sequence of SEQ ID NO: 27, and the        third polypeptide and the fourth polypeptide are a commonly        shared L chain of SEQ ID NO: 28;    -   (s) a bispecific antibody (Q499-z121/J327-z119/L404-k), wherein        the first polypeptide is an H chain consisting of the amino acid        sequence of SEQ ID NO: 20, the second polypeptide is an H chain        consisting of the amino acid sequence of SEQ ID NO: 25, and the        third polypeptide and the fourth polypeptide are a commonly        shared L chain of SEQ ID NO: 32;    -   (t) a bispecific antibody (Q499-z121/J339-z119/L377-k), wherein        the first polypeptide is an H chain consisting of the amino acid        sequence of SEQ ID NO: 20, the second polypeptide is an H chain        consisting of the amino acid sequence of SEQ ID NO: 26, and the        third polypeptide and the fourth polypeptide are a commonly        shared L chain of SEQ ID NO: 31; and    -   (u) a bispecific antibody (Q153-G4k/J142-G4h/L180-k), wherein        the first polypeptide is an H chain consisting of the amino acid        sequence of SEQ ID NO: 12, the second polypeptide is an H chain        consisting of the amino acid sequence of SEQ ID NO: 170, and the        third polypeptide and the fourth polypeptide are a commonly        shared L chain of SEQ ID NO: 171;        [33] a nucleic acid encoding the bispecific antibody of [32];        [34] a vector inserted with the nucleic acid of [33];        [35] a cell comprising the nucleic acid of [33] or the vector of        [34];        [36] a method for producing the bispecific antibody of [32] by        culturing the cell of [35];        [37] a pharmaceutical composition comprising the bispecific        antibody of [32], and a pharmaceutically acceptable carrier;        [38] the composition of [37], which is a pharmaceutical        composition used for prevention and/or treatment of bleeding, a        disease accompanying bleeding, or a disease caused by bleeding;        [39] the composition of [38], wherein the bleeding, the disease        accompanying bleeding, or the disease caused by bleeding is a        disease that develops and/or progresses due to a decrease or        deficiency in the activity of blood coagulation factor VIII        and/or activated blood coagulation factor VIII;        [40] the composition of [39], wherein the disease that develops        and/or progresses due to a decrease or deficiency in the        activity of blood coagulation factor VIII and/or activated blood        coagulation factor VIII is hemophilia A;        [41] the composition of [39], wherein the disease that develops        and/or progresses due to a decrease or deficiency in the        activity of blood coagulation factor VIII and/or activated blood        coagulation factor VIII is a disease showing emergence of an        inhibitor against blood coagulation factor VIII and/or activated        blood coagulation factor VIII;        [42] the composition of [39], wherein the disease that develops        and/or progresses due to a decrease or deficiency in the        activity of blood coagulation factor VIII and/or activated blood        coagulation factor VIII is acquired hemophilia;        [43] the composition of [39], wherein the disease that develops        and/or progresses due to a decrease in the activity of blood        coagulation factor VIII and/or activated blood coagulation        factor VIII is von Willebrand disease;        [44] a method for preventing and/or treating bleeding, a disease        accompanying bleeding, or a disease caused by bleeding, which        comprises the step of administering the bispecific antibody of        [32] or the composition of any one of [37] to [43];        [45] a kit for use in the prevention and/or treatment method of        [44], which comprises the bispecific antibody of [32], or the        composition of any one of [37] to [43];        [46] use of the bispecific antibody of [32] or the composition        of any one of [37] to [43] in the manufacture of an agent for        preventing and/or treating bleeding, a disease accompanying        bleeding, or a disease caused by bleeding; and        [47] the bispecific antibody of [32] or the composition of any        one of [37] to [43] for preventing and/or treating bleeding, a        disease accompanying bleeding, or a disease caused by bleeding.

Effects of the Invention

The present invention provides antibodies that recognize both an enzymeand its substrate, which are multispecific antigen-binding moleculeshaving a high activity of functionally substituting for F.VIII.Furthermore, the present invention provides antibodies that recognizeboth an enzyme and its substrate, which are multispecificantigen-binding molecules having a high activity of functionallysubstituting for F.VIII and a low F.Xase inhibitory action. Sincehumanized antibodies are generally thought to have high stability inblood and low immunogenicity, multispecific antibodies of the presentinvention may be very promising as pharmaceuticals.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 describes the F.Xase inhibitory action.

(a) F.VIIIa forms a complex with F.IXa (F.Xase) and activates F.X.

(b) A bispecific antibody binds to F.IXa and F.X and activates F.X.

(c) Both F.VIIIa and the bispecific antibody activate F.X withoutcompetition.

(d) Binding of the bispecific antibody to F.IXa and/or F.X inhibits theformation of the complex formed between F.Xase and F.X.

(e) Binding of the bispecific antibody to F.IXa and/or F.X inhibits theactivity of F.Xase.

FIG. 2 describes the screening. Approximately 200 types each of genesfor antibodies against human F.IXa and human F.X were produced, and theywere incorporated into animal cell expression vectors. 40,000 or morebispecific antibodies as a combination of an anti-F.IXa antibody andanti-F.X antibody were transiently expressed. F.Xa generation-promotingactivity and F.Xase inhibitory action were evaluated to screen forbispecific antibodies having a high F.Xa generation-promoting activityand a low F.Xase inhibitory action. Furthermore, by substituting aminoacids when necessary, prototype antibodies were produced.

FIG. 3 shows the F.Xa generation-promoting activities ofhA69-KQ/hB26-PF/hAL-AQ, Q1-G4k/J268-G4h/L45-k, Q1-G4k/J321-G4h/L45-k,Q31-z7/J326-z107/L2-k, and Q64-z55/J344-z107/L45-k. The concentrationsof the antibody solutions were 300, 30, and 3 μg/mL (the concentrationsafter mixing Human Factor IXa, Novact (registered trademark) M, HumanFactor X, and the antibody solution were 100, 10, and 1 μg/mL), theenzyme reaction and color development were performed for ten minutes and50 minutes, respectively. As a result, these antibodies showed a higherF.Xa generation-promoting activity compared to hA69-KQ/hB26-PF/hAL-AQdescribed in WO 2006/109592.

FIG. 4 shows the F.Xa generation-promoting activity ofhA69-KQ/hB26-PF/hAL-AQ, prototype antibodies, and modified antibodieswith amino acid substitutions. The concentrations of the antibodysolutions were 300, 30, and 3 μg/mL (the concentrations after mixingHuman Factor IXa, Novact (registered trademark) M, Human Factor X, andthe antibody solution were 100, 10, and 1 μg/mL), the enzyme reactionand color development were performed for two minutes and 20 minutes,respectively. As a result, these modified antibodies showed a higherF.Xa generation-promoting activity compared to the prototype antibodies.

FIG. 5 shows the F.Xase inhibitory action of hA69-KQ/hB26-PF/hAL-AQ,prototype antibodies, and modified antibodies with amino acidsubstitutions.

The figure shows the effects of hA69-KQ/hB26-PF/hAL-AQ,Q1-G4k/J268-G4h/L45-k, Q31-z7/J326-z107/L2-k, Q1-G4k/J321-G4h/L45-k,Q64-z55/J344-z107/L45-k, Q85-G4k/J268-G4h/L406-k,Q85-G4k/J321-G4h/L334-k, Q64-z7/J344-z107/L406-k,Q64-z7/J326-z107/L334-k, Q153-G4k/J142-G4h/L180-k,Q405-G4k/J232-G4h/L248-k, Q360-G4k/J232-G4h/L406-k,Q153-G4k/J232-G4h/L406-k, Q458-z106/J346-z107/L408-k,Q360-z118/J300-z107/L334-k, Q499-z118/J327-z107/L377-k,Q499-z121/J327-z119/L404-k, Q499-z121/J339-z119/L377-k,Q499-z118/J346-z107/L248-k, Q354-z106/J259-z107/L324-k,Q460-z121/J327-z119/L334-k, and Q499-z118/J327-z107/L334-k on F.Xactivation by F.IXa in the presence of F.VIIIa. The F.Xase inhibitoryactions of the antibodies are indicated as the value obtained bysubtracting the absorbance of the antibody-free reaction solution fromthe absorbance of the antibody-supplemented reaction solution. Theconcentrations of the antibody solutions were 300 and 30 μg/mL (theconcentrations after mixing Human Factor IXa, F.VIIIa, Human Factor X,and the antibody solution were 100 and 10 μg/mL), the enzyme reactionand color development were performed for six minutes and 14 minutes,respectively. The more positive the value of F.Xase inhibitory actionshown on the horizontal axis, the weaker the F.Xase inhibitory actionis. As a result, hA69-KQ/hB26-PF/hAL-AQ described in WO 2006/109592showed strong F.Xase inhibitory action. All of the antibodies of thepresent invention showed weaker F.Xase inhibitory action compared tohA69-KQ/hB26-PF/hAL-AQ, or did not show inhibitory action.

FIG. 6A shows the amino acid sequences of the prototype antibodies andthe modified antibodies with amino acid substitutions. When the sequencename is not indicated in the Ref column, the variable region sequence ofthe Name column is mentioned. A “- (hyphen)” is shown where an aminoacid is absent at the number by Kabat numbering. A “. (dot)” is shownwhere amino acid is the same when comparing the variable region of theName column and the Ref column, and the amino acid of the variableregion of the Name column is shown where the amino acids are different.Amino acids found to be important for improvement of F.Xageneration-promoting activity were indicated by framing them.

FIG. 6B is a continuation of FIG. 6A.

FIG. 6C is a continuation of FIG. 6B.

FIG. 6D is a continuation of FIG. 6C.

MODE FOR CARRYING OUT THE INVENTION

Multispecific antigen-binding molecules described herein comprise afirst antigen-binding site and a second antigen-binding site that canspecifically bind to at least two different types of antigens. While thefirst antigen-binding site and the second antigen-binding site are notparticularly limited as long as they have an activity to bind to F.IXand/or F.IXa, and F.X, respectively, examples include sites necessaryfor binding with antigens, such as antibodies, scaffold molecules(antibody-like molecules) or peptides, or fragments containing suchsites. Scaffold molecules are molecules that exhibit function by bindingto target molecules, and any polypeptide may be used as long as they areconformationally stable polypeptides that can bind to at least onetarget antigen. Examples of such polypeptides include antibody variableregions, fibronectin (WO 2002/032925), protein A domain (WO1995/001937), LDL receptor A domain (WO 2004/044011, WO 2005/040229),ankyrin (WO 2002/020565), and such, and also molecules described indocuments by Nygren et al. (Current Opinion in Structural Biology, 7:463-469 (1997); and Journal of Immunol Methods, 290: 3-28 (2004)), Binzet al. (Nature Biotech 23: 1257-1266 (2005)), and Hosse et al. (ProteinScience 15: 14-27 (2006)). Furthermore, as mentioned in Curr Opin MolTher. 2010 August; 12(4): 487-95 and Drugs. 2008; 68(7): 901-12, peptidemolecules that can bind to target antigens may be used.

Herein, multispecific antigen-binding molecules are not particularlylimited as long as they are molecules that can bind to at least twodifferent types of antigens, but examples include polypeptidescontaining the above-mentioned antigen-binding sites, such as antibodiesand scaffold molecules as well as their fragments, and aptamerscomprising nucleic acid molecules and peptides, and they may be singlemolecules or multimers thereof. Preferred multispecific antigen-bindingmolecules include multispecific antibodies that can bind specifically toat least two different antigens. Particularly preferred examples ofantibodies which have an activity of functionally substituting forF.VIII of the present invention include bispecific antibodies (BsAb)that can bind specifically to two different antigens (they may also becalled dual specific antibodies).

In the present invention, the term “commonly shared L chain” refers toan L chain that can link with two or more different H chains, and showbinding ability to each antigen. Herein, the term “different H chain(s)”preferably refers to H chains of antibodies against different antigens,but is not limited thereto, and also refers to H chains whose amino acidsequences are different from each other. Commonly shared L chain can beobtained, for example, according to the method described in WO2006/109592.

The multispecific antigen-binding molecules of the present invention(preferably bispecific antibodies) are antibodies having specificity totwo or more different antigens, or molecules comprising fragments ofsuch antibodies. The antibodies of the present invention are notparticularly limited, but are preferably monoclonal antibodies.Monoclonal antibodies used in the present invention include not onlymonoclonal antibodies derived from animals such as humans, mice, rats,hamsters, rabbits, sheep, camels, and monkeys, but also includeartificially modified gene recombinant antibodies such as chimericantibodies, humanized antibodies, and bispecific antibodies.

Furthermore, the L chains of an antibody which will become amultispecific antigen-binding molecule of the present invention may bedifferent, but preferably have commonly shared L chains.

Multispecific antigen-binding molecules of the present invention arepreferably recombinant antibodies produced using genetic recombinationtechniques (See, for example, Borrebaeck CAK and Larrick J W,THERAPEUTIC MONOCLONAL ANTIBODIES, Published in the United Kingdom byMACMILLAN PUBLISHERS LTD, 1990). Recombinant antibodies can be obtainedby cloning DNAs encoding antibodies from hybridomas orantibody-producing cells, such as sensitized lymphocytes, that produceantibodies, inserting them into suitable vectors, and then introducingthem into hosts (host cells) to produce the antibodies.

Furthermore, antibodies of the present invention may include not onlywhole antibodies but also antibody fragments and low-molecular-weightantibodies (minibodies), and modified antibodies.

For example, antibody fragments or minibodies include diabodies (Dbs),linear antibodies, and single chain antibody (hereinafter, also denotedas scFvs) molecules. Herein, an “Fv” fragment is defined as the smallestantibody fragment that comprises a complete antigen recognition site andbinding site.

An “Fv” fragment is a dimer (VH-VL dimer) in which an H chain variableregion (VH) and an L chain variable region (VL) are strongly linked bynon-covalent binding. The three complementarity determining regions(CDRs) of each of the variable regions interact with each other to forman antigen-binding site on the surface of the VH-VL dimer. Six CDRsconfer the antigen-binding site to an antibody. However, one variableregion (or half of the Fv comprising only three CDRs specific to anantigen) alone can recognize and bind to an antigen, though its affinityis lower than that of the entire binding site.

An Fab fragment (also called F(ab)) further comprises an L chainconstant region and an H chain constant region (CH1). An Fab′ fragmentdiffers from an Fab fragment in that it additionally comprises severalresidues derived from the carboxyl terminus of the H chain CH1 region,comprising one or more cysteines from the hinge region of the antibody.Fab′-SH refers to an Fab′ in which one or more cysteine residues of itsconstant region comprise a free thiol group. An F(ab′) fragment isproduced by cleavage of disulfide bonds between the cysteine residues inthe hinge region of F(ab′)₂ pepsin digest. Other chemically boundantibody fragments are also known to those skilled in the art.

Diabodies are bivalent minibodies constructed by gene fusion (Holliger,P. et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993); EP 404,097;WO 93/11161). Diabodies are dimers consisting of two polypeptide chains,in which each polypeptide chain comprises an L chain variable region(VL) and an H chain variable region (VH) linked with a linker shortenough to prevent association of these two domains within the samechain, for example, a linker of preferably 2 to 12 amino acids, morepreferably 3 to 10 amino acids, particularly about 5 amino acids. Thepolypeptide chain form a dimer since the linker between the VL and VHencoded on the same polypeptide is too short to form a single chainvariable region fragment. Therefore, diabodies comprise twoantigen-binding sites.

A single-chain antibody or an scFv antibody fragment comprises the VHand VL regions of an antibody, and these regions exist in a singlepolypeptide chain. In general, an Fv polypeptide further comprises apolypeptide linker between the VH and VL regions, and this enables anscFv to form a structure necessary for antigen binding (for a review onscFvs, see Pluckthun “The Pharmacology of Monoclonal Antibodies” Vol.113 (Rosenburg and Moore ed. (Springer Verlag, New York) pp. 269-315,1994). In the context of the present invention, linkers are notparticularly limited so long as they do not inhibit the expression ofthe antibody variable regions linked at their ends.

IgG-type bispecific antibodies can be secreted from hybrid hybridomas(quadromas) produced by fusing two kinds of hybridomas that produce IgGantibodies (Milstein C et al. Nature 1983, 305: 537-540). They can alsobe secreted by taking the L chain and H chain genes constituting the twokinds of IgGs of interest, a total of four kinds of genes, andintroducing them into cells to coexpress the genes.

In this case, by introducing suitable amino acid substitutions to theCH3 regions of the H chains, IgGs having a heterogeneous combination ofH chains can be preferentially secreted (Ridgway J B et al. ProteinEngineering 1996, 9: 617-621; Merchant A M et al. Nature Biotechnology1998, 16: 677-681; WO 2006/106905; Davis J H et al. Protein Eng Des Sel.2010, 4: 195-202).

Regarding the L chains, since diversity of L chain variable regions islower than that of H chain variable regions, commonly shared L chainsthat can confer binding ability to both H chains may be obtained. Theantibodies of the present invention comprise commonly shared L chains.Bispecific IgGs can be efficiently expressed by introducing the genes ofthe commonly shared L chain and both H chains into cells.

Bispecific antibodies may be produced by chemically crosslinking Fab′s.Bispecific F(ab′)₂ can be produced, for example, by preparing Fab′ froman antibody, using it to produce a maleimidized Fab′ withortho-phenylenedi-maleimide (o-PDM), and then reacting this with Fab′prepared from another antibody to crosslink Fab′s derived from differentantibodies (Keler T et al. Cancer Research 1997, 57: 4008-4014). Themethod of chemically linking an Fab′-thionitrobenzoic acid (TNB)derivative and an antibody fragment such as Fab′-thiol (SH) is alsoknown (Brennan Metal. Science 1985, 229: 81-83).

Instead of a chemical crosslink, a leucine zipper derived from Fos andJun may also be used. Preferential formation of heterodimers by Fos andJun is utilized, even though they also form homodimers. Fab′ to whichFos leucine zipper is added, and another Fab′ to which Jun leucinezipper is added are expressed and prepared. Monomeric Fab′-Fos andFab′-Jun reduced under mild conditions are mixed and reacted to formbispecific F(ab′)₂ (Kostelny S A et al. J. of Immunology, 1992, 148:1547-53). This method can be applied not only to Fab′s but also toscFvs, Fvs, and such.

Furthermore, bispecific antibodies including sc(Fv)₂ such as IgG-scFv(Protein Eng Des Sel. 2010 April; 23(4): 221-8) and BiTE (Drug DiscovToday. 2005 Sep. 15; 10(18): 1237-44.), DVD-Ig (Nat. Biotechnol. 2007November; 25(11): 1290-7. Epub 2007 Oct. 14.; and MAbs. 2009 July; 1(4):339-47. Epub 2009 July 10.), and also others (IDrugs 2010, 13: 698-700)including two-in-one antibodies (Science. 2009 Mar. 20; 323(5921):1610-4; and Immunotherapy. 2009 September; 1(5): 749-51.), Tri-Fab,tandem scFv, and diabodies are known (MAbs. 2009 November; 1(6):539-547). In addition, even when using molecular forms such as scFv-Fcand scaffold-Fc, bispecific antibodies can be produced efficiently bypreferentially secreting a heterologous combination of Fcs (Ridgway J Bet al., Protein Engineering 1996, 9: 617-621; Merchant A M et al. NatureBiotechnology 1998, 16: 677-681; WO 2006/106905; and Davis J H et al.,Protein Eng Des Sel. 2010, 4: 195-202.).

A bispecific antibody may also be produced using a diabody. A bispecificdiabody is a heterodimer of two cross-over scFv fragments. Morespecifically, it is produced by forming a heterodimer using VH(A)-VL(B)and VH(B)-VL(A) prepared by linking VHs and VLs derived from two kindsof antibodies, A and B, using a relatively short linker of about 5residues (Holliger P et al. Proc Natl. Acad. Sci. USA 1993, 90:6444-6448).

The desired structure can be achieved by linking the two scFvs with aflexible and relatively long linker comprising about 15 residues (singlechain diabody: Kipriyanov S M et al. J. of Molecular Biology. 1999, 293:41-56), and conducting appropriate amino acid substitutions(knobs-into-holes: Zhu Z et al. Protein Science. 1997, 6: 781-788; VH/VLinterface engineering: Igawa T et al. Protein Eng Des Sel. 2010, 8:667-77).

An sc(Fv)₂ that can be produced by linking two types of scFvs with aflexible and relatively long linker, comprising about 15 residues, mayalso be a bispecific antibody (Mallender W D et al. J. of BiologicalChemistry, 1994, 269: 199-206).

Examples of modified antibodies include antibodies linked to variousmolecules such as polyethylene glycol (PEG). The antibodies of thepresent invention include such modified antibodies. In the context ofthe present invention, the substance to which the modified antibodiesare linked is not limited. Such modified antibodies can be obtained bychemically modifying obtained antibodies. Such methods are wellestablished in the art.

The antibodies of the present invention include human antibodies, mouseantibodies, rat antibodies, or such, and their origins are not limited.They may also be genetically modified antibodies, such as chimeric orhumanized antibodies.

Methods for obtaining human antibodies are known in the art. Forexample, transgenic animals carrying the entire repertoire of humanantibody genes can be immunized with desired antigens to obtain desiredhuman antibodies (see International Patent Application WO 93/12227, WO92/03918, WO 94/02602, WO 94/25585, WO 96/34096, and WO 96/33735).

Genetically modified antibodies can also be produced using knownmethods. Specifically, for example, chimeric antibodies may comprise Hchain and L chain variable regions of an immunized animal antibody, andH chain and L chain constant regions of a human antibody. Chimericantibodies can be obtained by linking DNAs encoding the variable regionsof the antibody derived from the immunized animal, with DNAs encodingthe constant regions of a human antibody, inserting this into anexpression vector, and then introducing it into host cells to producethe antibodies.

Humanized antibodies are modified antibodies often referred to as“reshaped” human antibodies. A humanized antibody is constructed bytransferring the CDRs of an antibody derived from an immunized animal tothe complementarity determining regions of a human antibody.Conventional genetic recombination techniques for such purposes areknown (see European Patent Application Publication No. EP 239400;International Publication No. WO 96/02576; Sato K et al., CancerResearch 1993, 53: 851-856; International Publication No. WO 99/51743).

The multispecific antigen-binding molecules of the present invention arethose that recognize F.IX and/or F.IXa, and F.X, and functionallysubstitute for cofactor function of F.VIII, and characterized in thatthe molecules have a higher F.Xa generation-promoting activity comparedto hA69-KQ/hB26-PF/hAL-AQ (described in WO 2006/109592) which is knownas a bispecific antibody that functionally substitutes for F.VIII.Furthermore, antibodies of the present invention usually have astructure which comprises a variable region of an anti-F.IXa antibodyand a variable region of an anti-F.X antibody.

More specifically, the present invention provides a multispecificantigen-binding molecule that functionally substitutes for F.VIII, whichcomprises a first antigen-binding site that recognizes F.IX and/or F.IXaand a second antigen-binding site that recognizes F.X, wherein thefunction that substitutes for the function of F.VIII is caused by ahigher F.Xa generation-promoting activity compared to the activity ofthe bispecific antibody (hA69-KQ/hB26-PF/hAL-AQ) which comprises Hchains consisting of SEQ ID NOs: 165 and 166, and a commonly shared Lchain consisting of SEQ ID NO: 167.

A multispecific antigen-binding molecule of the present inventioncomprises a first polypeptide and a third polypeptide comprising anantigen-binding site that recognizes F.IX and/or F.IXa, and a secondpolypeptide and a fourth polypeptide comprising an antigen-binding sitethat recognizes F.X. The first polypeptide and the third polypeptide,and the second polypeptide and the fourth polypeptide each include theantigen-binding site of the antibody H chain and the antigen-bindingsite of the antibody L chain.

For example, in a multispecific antigen-binding molecule of the presentinvention, the first polypeptide and the third polypeptide include anantigen-binding site of an H chain and L chain of an antibody againstF.IX or F.IXa, respectively; and the second polypeptide and the fourthpolypeptide comprise an antigen-binding site of an H chain and L chainof an antibody against F.X, respectively.

At this time, the antigen-binding sites of the antibody L chain includedin the first polypeptide and the third polypeptide, and the secondpolypeptide and the fourth polypeptide may be commonly shared L chains.

A polypeptide comprising an antigen-binding site of an antibody L chainin the present invention is preferably a polypeptide which comprises allor a part of the sequence of the antibody L chain which binds to F.IX,F.IXa and/or F.X.

Preferred embodiments of the antigen-binding site of the firstpolypeptide of an antibody of the present invention specifically includeantigen-binding sites comprising the amino acid sequences of:

Q1 H chain each CDR1, 2, and 3 sequence (SEQ ID NOs: 75, 76, and 77,respectively);Q31 H chain each CDR1, 2, and 3 sequences (SEQ ID NOs: 78, 79, and 80,respectively);Q64 H chain each CDR1, 2, and 3 sequence (SEQ ID NOs: 81, 82, and 83,respectively);Q85 H chain each CDR1, 2, and 3 sequence (SEQ ID NOs: 84, 85, and 86,respectively);Q153 H chain each CDR1, 2, and 3 sequence (SEQ ID NOs: 87, 88, and 89,respectively);Q354 H chain each CDR1, 2, and 3 sequence (SEQ ID NOs: 90, 91, and 92,respectively);Q360 H chain each CDR1, 2, and 3 sequence (SEQ ID NOs: 93, 94, and 95,respectively);Q405 H chain each CDR1, 2, and 3 sequence (SEQ ID NOs: 96, 97, and 98,respectively);Q458 H chain each CDR1, 2, and 3 sequence (SEQ ID NOs: 99, 100, and 101,respectively);Q460 H chain each CDR1, 2, and 3 sequence (SEQ ID NOs: 102, 103, and104, respectively); andQ499 H chain each CDR1, 2, and 3 sequence (SEQ ID NOs: 105, 106, and107, respectively) mentioned in the later-described Examples, orantigen-binding sites that are functionally equivalent to them.

Preferred embodiments of the antigen-binding site of a secondpolypeptide specifically include, for example, antigen-binding sitescomprising the amino acid sequences of:

J232 H chain each CDR1, 2, and 3 sequence (SEQ ID NOs: 108, 109, and110, respectively);J259 H chain each CDR1, 2, and 3 sequence (SEQ ID NOs: 111, 112, and113, respectively);J268 H chain each CDR1, 2, and 3 sequence (SEQ ID NOs: 114, 115, and116, respectively);J300 H chain each CDR1, 2, and 3 sequence (SEQ ID NOs: 117, 118, and119, respectively);J321 H chain each CDR1, 2, and 3 sequence (SEQ ID NOs: 120, 121, and122, respectively);J326 H chain each CDR1, 2, and 3 sequence (SEQ ID NOs: 123, 124, and125, respectively);J327 H chain each CDR1, 2, and 3 sequence (SEQ ID NOs: 126, 127, and128, respectively);J339 H chain each CDR1, 2, and 3 sequence (SEQ ID NOs: 129, 130, and131, respectively);J344 H chain each CDR1, 2, and 3 sequence (SEQ ID NOs: 132, 133, and134, respectively);J346 H chain each CDR1, 2, and 3 sequence (SEQ ID NOs: 135, 136, and137, respectively); andJ142 H chain each CDR1, 2, and 3 sequence (SEQ ID NOs: 174, 175, and176, respectively) mentioned in the later-described Examples, orantigen-binding sites that are functionally equivalent to them.

More specifically, the present invention provides multispecificantigen-binding molecules, wherein the antigen-binding site of the firstpolypeptide comprises an antigen-binding site which comprises H chainCDRs consisting of any one of the amino acid sequences selected from thefollowing (a1) to (a11), or an antigen-binding site functionallyequivalent thereto, and the antigen-binding site of the secondpolypeptide comprises an antigen-binding site which comprises H chainCDRs consisting of any one of the amino acid sequences selected from thefollowing (b1) to (b11), or an antigen-binding site functionallyequivalent thereto:

-   -   (a1) an antigen-binding site comprising an H chain CDR 1, 2, and        3 amino acid sequences of SEQ ID NOs: 75, 76, and 77 (H chain        CDRs of Q1), respectively;    -   (a2) an antigen-binding site comprising an H chain CDR 1, 2, and        3 amino acid sequences of SEQ ID NOs: 78, 79, and 80 (H chain        CDRs of Q31), respectively;    -   (a3) an antigen-binding site comprising an H chain CDR 1, 2, and        3 amino acid sequences of SEQ ID NOs: 81, 82, and 83 (H chain        CDRs of Q64), respectively;    -   (a4) an antigen-binding site comprising an H chain CDR 1, 2, and        3 amino acid sequences of SEQ ID NOs: 84, 85, and 86 (H chain        CDRs of Q85), respectively;    -   (a5) an antigen-binding site comprising the H chain CDR 1, 2,        and 3 amino acid sequences of SEQ ID NOs: 87, 88, and 89 (H        chain CDRs of Q153), respectively;    -   (a6) an antigen-binding site comprising an H chain CDR 1, 2, and        3 amino acid sequences of SEQ ID NOs: 90, 91, and 92 (H chain        CDRs of Q354), respectively;    -   (a7) an antigen-binding site comprising the H chain CDR 1, 2,        and 3 amino acid sequences of SEQ ID NOs: 93, 94, and 95 (H        chain CDRs of Q360), respectively;    -   (a8) an antigen-binding site comprising the of H chain CDR 1, 2,        and 3 amino acid sequences of SEQ ID NOs: 96, 97, and 98 (H        chain CDRs of Q405), respectively;    -   (a9) an antigen-binding site comprising an H chain CDR 1, 2, and        3 amino acid sequences of SEQ ID NOs: 99, 100, and 101 (H chain        CDRs of Q458), respectively;    -   (a10) an antigen-binding site comprising an H chain CDR 1, 2,        and 3 amino acid sequences of SEQ ID NOs: 102, 103, and 104 (H        chain CDRs of Q460), respectively;    -   (a11) an antigen-binding site comprising an H chain CDR 1, 2,        and 3 amino acid sequences of SEQ ID NOs: 105, 106, and 107 (H        chain CDRs of Q499), respectively;    -   (b1) an antigen-binding site comprising an H chain CDR 1, 2, and        3 amino acid sequences of SEQ ID NOs: 108, 109, and 110 (H chain        CDRs of J232), respectively;    -   (b2) an antigen-binding site comprising an H chain CDR 1, 2, and        3 amino acid sequences of SEQ ID NOs: 111, 112, and 113 (H chain        CDRs of J259), respectively;    -   (b3) an antigen-binding site comprising an H chain CDR 1, 2, and        3 amino acid sequences of SEQ ID NOs: 114, 115, and 116 (H chain        CDRs of J268), respectively;    -   (b4) an antigen-binding site comprising an H chain CDR 1, 2, and        3 amino acid sequences of SEQ ID NOs: 117, 118, and 119 (H chain        CDRs of J300), respectively;    -   (b5) an antigen-binding site comprising an H chain CDR 1, 2, and        3 amino acid sequences of SEQ ID NOs: 120, 121, and 122 (H chain        CDRs of J321), respectively;    -   (b6) an antigen-binding site comprising the H chain CDR 1, 2,        and 3 amino acid sequences of SEQ ID NOs: 123, 124, and 125 (H        chain CDRs of J326), respectively;    -   (b7) an antigen-binding site comprising an H chain CDR 1, 2, and        3 amino acid sequences of SEQ ID NOs: 126, 127, and 128 (H chain        CDRs of J327), respectively;    -   (b8) an antigen-binding site comprising an H chain CDR 1, 2, and        3 amino acid sequences of SEQ ID NOs: 129, 130, and 131 (H chain        CDRs of J339), respectively;    -   (b9) an antigen-binding site comprising an H chain CDR 1, 2, and        3 amino acid sequences of SEQ ID NOs: 132, 133, and 134 (H chain        CDRs of J344), respectively;    -   (b10) an antigen-binding site comprising an H chain CDR 1, 2,        and 3 amino acid sequences of SEQ ID NOs: 135, 136, and 137 (H        chain CDRs of J346), respectively; and    -   (b11) an antigen-binding site comprising an H chain CDR 1, 2,        and 3 amino acid sequences of SEQ ID NOs: 174, 175, and 176 (H        chain CDRs of J142), respectively.

Preferred embodiments of the antigen-binding site of the third andfourth polypeptides specifically include, for example, antigen-bindingsites comprising the amino acid sequences of:

L2 L chain each CDR1, 2, and 3 sequence (SEQ ID NOs: 138, 139, and 140,respectively);L45 L chain each CDR1, 2, and 3 sequence (SEQ ID NOs: 141, 142, and 143,respectively);L248 L chain each CDR1, 2, and 3 sequence (SEQ ID NOs: 144, 145, and146, respectively);L324 L chain each CDR1, 2, and 3 sequence (SEQ ID NOs: 147, 148, and149, respectively);L334 L chain each CDR1, 2, and 3 sequence (SEQ ID NOs: 150, 151, and152, respectively);L377 L chain each CDR1, 2, and 3 sequence (SEQ ID NOs: 153, 154, and155, respectively);L404 L chain each CDR1, 2, and 3 sequence (SEQ ID NOs: 156, 157, and158, respectively);L406 L chain each CDR1, 2, and 3 sequence (SEQ ID NOs: 159, 160, and161, respectively);L408 L chain each CDR1, 2, and 3 sequence (SEQ ID NOs: 162, 163, and164, respectively); andL180 L chain each CDR1, 2, and 3 sequence (SEQ ID NOs: 177, 178, and179, respectively) mentioned in the later-described Examples, orantigen-binding sites that are functionally equivalent to them.

More specifically, the present invention provides multispecificantigen-binding molecules, wherein the antigen-binding sites included inthe third polypeptide and the fourth polypeptide comprise anantigen-binding site which comprises L chain CDRs consisting of any oneof the amino acid sequences selected from the following (c1) to (c10),or an antigen-binding site functionally equivalent thereto:

-   -   (c1) an antigen-binding site comprising an L chain CDR1, 2, and        3 amino acid sequences of SEQ ID NOs: 138, 139, and 140 (L chain        CDR of L2), respectively;    -   (c2) an antigen-binding site comprising an L chain CDR1, 2, and        3 amino acid sequences of SEQ ID NOs: 141, 142, and 143 (L chain        CDR of L45), respectively;    -   (c3) an antigen-binding site comprising an L chain CDR1, 2, and        3 amino acid sequences of SEQ ID NOs: 144, 145, and 146 (L chain        CDR of L248), respectively;    -   (c4) an antigen-binding site comprising an L chain CDR1, 2, and        3 amino acid sequences of SEQ ID NOs: 147, 148, and 149 (L chain        CDR of L324), respectively;    -   (c5) an antigen-binding site comprising an L chain CDR1, 2, and        3 amino acid sequences of SEQ ID NOs: 150, 151, and 152 (L chain        CDR of L334), respectively;    -   (c6) an antigen-binding site comprising an L chain CDR1, 2, and        3 amino acid sequences of SEQ ID NOs: 153, 154, and 155 (L chain        CDR of L377), respectively;    -   (c7) an antigen-binding site comprising an L chain CDR1, 2, and        3 amino acid sequences of SEQ ID NOs: 156, 157, and 158 (L chain        CDR of L404), respectively;    -   (c8) an antigen-binding site comprising an L chain CDR1, 2, and        3 amino acid sequences of SEQ ID NOs: 159, 160, and 161 (L chain        CDR of L406), respectively;    -   (c9) an antigen-binding site comprising an L chain CDR1, 2, and        3 amino acid sequences of SEQ ID NOs: 137, 138, and 139 (L chain        CDR of L408), respectively; and    -   (c10) an antigen-binding site comprising an L chain CDR1, 2, and        3 amino acid sequences of SEQ ID NOs: 177, 178, and 179 (L chain        CDR of L180), respectively.

The amino acid sequences of the H chain variable regions of Q1, Q31,Q64, Q85, Q153, Q354, Q360, Q405, Q458, Q460, and Q499 of the presentinvention are indicated by the following SEQ ID NOs, respectively.

Q1: SEQ ID NO: 35

Q31: SEQ ID NO: 36

Q64: SEQ ID NO: 37

Q85: SEQ ID NO: 38

Q153: SEQ ID NO: 39

Q354: SEQ ID NO: 40

Q360: SEQ ID NO: 41

Q405: SEQ ID NO: 42

Q458: SEQ ID NO: 43

Q460: SEQ ID NO: 44

Q499: SEQ ID NO: 45

The amino acid sequences of the H chain variable regions of J232, J259,J268, J300, J321, J326, J327, J339, J344, J346, and J142 of the presentinvention are indicated by the following SEQ ID NOs, respectively.

J232: SEQ ID NO: 46

J259: SEQ ID NO: 47

J268: SEQ ID NO: 48

J300: SEQ ID NO: 49

J321: SEQ ID NO: 50

J326: SEQ ID NO: 51

J327: SEQ ID NO: 52

J339: SEQ ID NO: 53

J344: SEQ ID NO: 54

J346: SEQ ID NO: 55

J142: SEQ ID NO: 172

More specifically, the present invention provides multispecificantigen-binding molecules, wherein the antigen-binding site of the firstpolypeptide comprises an antigen-binding site which comprises an H chainvariable region consisting of any one of the amino acid sequencesselected from the following (a1) to (a11), or an antigen-binding sitefunctionally equivalent thereto, and the antigen-binding site of thesecond polypeptide comprises an antigen-binding site which comprises anH chain variable region consisting of any one of the amino acidsequences selected from the following (b1) to (b11), or anantigen-binding site functionally equivalent thereto:

-   -   (a1) an antigen-binding site comprising an H chain variable        region amino acid sequence of SEQ ID NO: 35 (H chain variable        region of Q1);    -   (a2) an antigen-binding site comprising an H chain variable        region amino acid sequence of SEQ ID NO: 36 (H chain variable        region of Q31);    -   (a3) an antigen-binding site comprising an H chain variable        region amino acid sequence of SEQ ID NO: 37 (H chain variable        region of Q1);    -   (a4) an antigen-binding site comprising an H chain variable        region amino acid sequence of SEQ ID NO: 38 (H chain variable        region of Q85);    -   (a5) an antigen-binding site comprising an H chain variable        region amino acid sequence of SEQ ID NO: 39 (H chain variable        region of Q153);    -   (a6) an antigen-binding site comprising an H chain variable        region amino acid sequence of SEQ ID NO: 40 (H chain variable        region of Q354);    -   (a7) an antigen-binding site comprising an H chain variable        region amino acid sequence of SEQ ID NO: 41 (H chain variable        region of Q360);    -   (a8) an antigen-binding site comprising an H chain variable        region amino acid sequence of SEQ ID NO: 42 (H chain variable        region of Q405);    -   (a9) an antigen-binding site comprising an H chain variable        region amino acid sequence of SEQ ID NO: 43 (H chain variable        region of Q458);    -   (a10) an antigen-binding site comprising an H chain variable        region amino acid sequence of SEQ ID NO: 44 (H chain variable        region of Q460);    -   (a11) an antigen-binding site comprising an H chain variable        region amino acid sequence of SEQ ID NO: 45 (H chain variable        region of Q499);    -   (b1) an antigen-binding site comprising an H chain variable        region amino acid sequence of SEQ ID NO: 46 (H chain variable        region of J232);    -   (b2) an antigen-binding site comprising an H chain variable        region amino acid sequence of SEQ ID NO: 47 (H chain variable        region of J259);    -   (b3) an antigen-binding site comprising an H chain variable        region amino acid sequence of SEQ ID NO: 48 (H chain variable        region of J268);    -   (b4) an antigen-binding site comprising an H chain variable        region amino acid sequence of SEQ ID NO: 49 (H chain variable        region of J300);    -   (b5) an antigen-binding site comprising an H chain variable        region amino acid sequence of SEQ ID NO: 50 (H chain variable        region of J321);    -   (b6) an antigen-binding site comprising an H chain variable        region amino acid sequence of SEQ ID NO: 51 (H chain variable        region of J326);    -   (b7) an antigen-binding site comprising an H chain variable        region amino acid sequence of SEQ ID NO: 52 (H chain variable        region of J327);    -   (b8) an antigen-binding site comprising an H chain variable        region amino acid sequence of SEQ ID NO: 53 (H chain variable        region of J339);    -   (b9) an antigen-binding site comprising an H chain variable        region amino acid sequence of SEQ ID NO: 54 (H chain variable        region of J344);    -   (b10) an antigen-binding site comprising an H chain variable        region amino acid sequence of SEQ ID NO: 55 (H chain variable        region of J346); and    -   (b11) an antigen-binding site comprising an H chain variable        region amino acid sequence of SEQ ID NO: 172 (H chain variable        region of J142).

In addition, the amino acid sequences of the L chain variable regions ofL2, L45, L248, L324, L334, L377, L404, L406, L408, and L180 of thepresent invention are indicated by the following SEQ ID NOs,respectively.

L2: SEQ ID NO: 56

L45: SEQ ID NO: 57

L248: SEQ ID NO: 58

L324: SEQ ID NO: 59

L334: SEQ ID NO: 60

L377: SEQ ID NO: 61

L404: SEQ ID NO: 62

L406: SEQ ID NO: 63

L408: SEQ ID NO: 64

L180: SEQ ID NO: 173

More specifically, the present invention provides multispecificantigen-binding molecules, wherein the antigen-binding sites included inthe third polypeptide and the fourth polypeptide comprise anantigen-binding site which comprises an L chain variable regionconsisting of any one of the amino acid sequences selected from thefollowing (c1) to (c10), or an antigen-binding site functionallyequivalent thereto:

-   -   (c1) an antigen-binding site comprising an L chain variable        region amino acid sequence of SEQ ID NO: 56 (L chain variable        region of L2);    -   (c2) an antigen-binding site comprising an L chain variable        region amino acid sequence of SEQ ID NO: 57 (L chain variable        region of L45);    -   (c3) an antigen-binding site comprising an L chain variable        region amino acid sequence of SEQ ID NO: 58 (L chain variable        region of L248);    -   (c4) an antigen-binding site comprising an L chain variable        region amino acid sequence of SEQ ID NO: 59 (L chain variable        region of L324);    -   (c5) an antigen-binding site comprising an L chain variable        region amino acid sequence of SEQ ID NO: 60 (L chain variable        region of L334);    -   (c6) an antigen-binding site comprising an L chain variable        region amino acid sequence of SEQ ID NO: 61 (L chain variable        region of L377);    -   (c7) an antigen-binding site comprising an L chain variable        region amino acid sequence of SEQ ID NO: 62 (L chain variable        region of L404);    -   (c8) an antigen-binding site comprising an L chain variable        region amino acid sequence of SEQ ID NO: 63 (L chain variable        region of L406);    -   (c9) an antigen-binding site comprising an L chain variable        region amino acid sequence of SEQ ID NO: 64 (L chain variable        region of L408); and    -   (c10) an antigen-binding site comprising an L chain variable        region amino acid sequence of SEQ ID NO: 173 (L chain variable        region of L180).

The amino acid sequences of CDR1 to 3 and FR1 to 4 in each of thesequences are as described in FIGS. 3A to D

When producing a full-length antibody using the variable regionsdisclosed in the present invention, without particular limitations,constant regions well known to those skilled in the art may be used. Forexample, constant regions described in “Sequences of proteins ofimmunological interest”, (1991), U.S. Department of Health and HumanServices. Public Health Service National Institutes of Health, or “Anefficient route to human bispecific IgG”, (1998). Nature Biotechnologyvol. 16, 677-681 can be used. Preferred examples of the antibodyconstant regions of the present invention include the constant regionsof IgG antibodies. When using the constant region of an IgG antibody,its type is not limited, and a constant region of IgG subclass such asIgG1, IgG2, IgG3, or IgG4 may be used. Furthermore, amino acid mutationsmay be introduced into the constant region of these IgG subclasses.Amino acid mutations to be introduced may be, for example, those thatenhance or decrease binding to Fcγ receptors (Proc Natl Acad Sci USA.2006 Mar. 14; 103(11): 4005-10; and MAbs. 2009 November; 1(6): 572-9),or enhance or decrease binding to FcRn (J Biol. Chem. 2001 Mar. 2;276(9): 6591-604; Int Immunol. 2006 December; 18(12): 1759-69; and JBiol Chem. 2006 Aug. 18; 281(33): 23514-24), but are not limitedthereto. Two types of H chains must be heterologously associated toproduce a bispecific antibody. The knobs-into-holes technology (JImmunol Methods. 2001 Feb. 1; 248(1-2): 7-15; and J Biol Chem. 2010 Jul.2; 285(27): 20850-9), the electrostatic repulsion technology (WO2006/106905), the SEEDbody technology (Protein Eng Des Sel. 2010 April;23(4): 195-202), and such may be used for heterologous association oftwo types of H chains via a CH3 domain. Furthermore, the antibodies ofthe present invention may be those with a modified or deficient sugarchain. Examples of antibodies having modified sugar chains includeglycosylation-engineered antibodies (such as WO 99/54342), antibodieswith defucosylated sugar chains (WO 00/61739, WO 02/31140, WO2006/067847, WO 2006/067913, etc.), and antibodies having a sugar chainwith bisecting GlcNAc (such as WO 02/79255). Known examples of methodsfor producing sugar chain-deficient IgG antibodies include the method ofintroducing a mutation to asparagine at position 297 in the EU numbering(J Clin Pharmacol. 2010 May; 50(5): 494-506), and the method ofproducing IgG using Escherichia coli (J Immunol Methods. 2002 May 1;263(1-2): 133-47; and J Biol. Chem. 2010 Jul. 2; 285(27): 20850-9).Furthermore, heterogeneity accompanying deletion of C-terminal lysine inIgG, and heterogeneity accompanying mispairing of disulfide bonds in thehinge region of IgG2 can be decreased by introducing amino aciddeletions/substitutions (WO 2009/041613).

The present invention provides, for example, multispecificantigen-binding molecules, wherein the first and second polypeptidescomprise an antibody H chain constant region, and the third and fourthpolypeptides comprise an antibody L chain constant region.

Furthermore, the present invention provides multispecificantigen-binding molecules, wherein the first polypeptide comprises anantibody H chain constant region consisting of any one of the amino acidsequences selected from the group consisting of the following (d1) to(d6) or the group consisting of the following (d7) to (d9), and thesecond polypeptide comprises an antibody H chain constant regionconsisting of any one of the amino acid sequences selected from a groupdifferent from that of the above-mentioned first polypeptide:

-   -   (d1) an H chain constant region of SEQ ID NO: 65 (G4k);    -   (d2) an H chain constant region of SEQ ID NO: 66 (z7);    -   (d3) an H chain constant region of SEQ ID NO: 67 (z55);    -   (d4) an H chain constant region of SEQ ID NO: 68 (z106);    -   (d5) an H chain constant region of SEQ ID NO: 69 (z118);    -   (d6) an H chain constant region of SEQ ID NO: 70 (z121);    -   (d7) an H chain constant region of SEQ ID NO: 71 (G4h);    -   (d8) an H chain constant region of SEQ ID NO: 72 (z107); and    -   (d9) an H chain constant region of SEQ ID NO: 73 (z119).

Furthermore, the present invention provides a multispecificantigen-binding molecule, wherein the third and fourth polypeptidescomprise an antibody L chain constant region consisting of the followingamino acid sequence of:

(e) an L chain constant region of SEQ ID NO: 74 (k).

In the present invention, the phrase “functionally substitute forF.VIII” means that F.IX and/or F.IXa, and F.X is recognized, andactivation of F.X is promoted (F.Xa generation is promoted).

In the present invention, “F.Xa generation-promoting activity” can beconfirmed by evaluating the multispecific antigen-binding molecules ofthe present invention using, for example, a measurement systemcomprising F.XIa (F.IX activating enzyme), F.IX, F.X, F syntheticsubstrate S-2222 (synthetic substrate of F.Xa), and phospholipids. Thismeasurement system shows the correlation between the severity of thedisease and clinical symptoms in hemophilia A cases (Rosen S, AnderssonM, Blomba{umlaut over ( )}ck M et al. Clinical applications of achromogenic substrate method for determination of FVIII activity. ThrombHaemost 1985, 54: 811-23). That is, in the present measurement system,test substances that show higher F.Xa generation-promoting activity areexpected to show better hemostatic effects against bleeding episodes inhemophilia A. With these results, if a multispecific antigen-bindingmolecule having activity of functionally substituting for F.VIII is amolecule having a higher activity than hA69-KQ/hB26-PF/hAL-AQ, it mayyield excellent blood coagulation-promoting activity, and excellenteffects may be obtained as a pharmaceutical component for preventingand/or treating bleeding, a disease accompanying bleeding, or a diseasecaused by bleeding. To obtain excellent effects as the above-mentionedpharmaceutical component, for example, F.Xa generation-promotingactivity measured under the conditions described in [Example 2] ispreferably not less than that of hA69-KQ/hB26-PF/hAL-AQ, and inparticular, the activity is more preferably the same as or not less thanthat of Q153-G4k/J142-G4h/L180-k. Herein, the “F.Xa generation-promotingactivity” is the value obtained by subtracting the change in absorbanceupon 20 minutes in a solvent from the change in absorbance upon 20minutes in an antibody solution.

A preferred embodiment of the present invention is a multispecificantibody that functionally substitutes for F.VIII, which recognizes F.IXand/or F.IXa, and F.X.

The above-mentioned multispecific antibodies of the present inventionare preferably antibodies which comprise H chain CDRs of anti-F.IX/F.IXaantibodies or CDRs functionally equivalent to them, and H chain CDRs ofanti-F.X antibodies or CDRs functionally equivalent to them.

Furthermore, the antibodies of the present invention are preferablyantibodies comprising an antigen-binding site having:

H chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs 75, 76, and77 (H chain CDRs of Q1), respectively;H chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs 78, 79, and80 (H chain CDRs of Q31), respectively;H chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs 81, 82, and83 (H chain CDRs of Q64), respectively;H chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs 84, 85, and86 (H chain CDRs of Q85), respectively;H chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs 87, 88, and89 (H chain CDRs of Q153), respectively;H chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs 90, 91, and92 (H chain CDRs of Q354), respectively;H chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs 93, 94, and95 (H chain CDRs of Q360), respectively;H chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs 96, 97, and98 (H chain CDRs of Q405), respectively;H chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs 99, 100, and101 (H chain CDRs of Q458), respectively;H chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs 102, 103, and104 (H chain CDRs of Q460), respectively; orH chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs 105, 106, and107 (H chain CDRs of Q499), respectively,in an anti-F.IX/IXa antibody, or an antigen-binding site functionallyequivalent to it, and an antigen-binding site comprising:H chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs 108, 109, and110 (H chain CDRs of J232), respectively;H chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs 111, 112, and113 (H chain CDRs of J259), respectively;H chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs 114, 115, and116 (H chain CDRs of J268), respectively;H chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs 117, 118, and119 (H chain CDRs of J300), respectively;H chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs 120, 121, and122 (H chain CDRs of J321), respectively;H chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs 123, 124, and125 (H chain CDRs of J326), respectively;H chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs 126, 127, and128 (H chain CDRs of J327), respectively;H chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs 129, 130, and131 (H chain CDRs of J339), respectively;H chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs 132, 133, and134 (H chain CDRs of J334), respectively;the amino acid sequences of H chain CDR 1, 2, and 3 amino acid sequencesof SEQ ID NOs 135, 136, and 137 (H chain CDRs of J346), respectively; orH chain CDR 1, 2, and 3 amino acid sequences of SEQ ID NOs 174, 175, and176 (H chain CDRs of J142), respectively,in an anti-F.X antibody, or an antigen-binding site functionallyequivalent to it.

In the present invention, “antigen-binding sites are functionallyequivalent” means that the activities of functionally substituting forF.VIII possessed by the multispecific antigen-binding molecules havingthe antigen-binding sites are equivalent.

In the present invention, the term “equivalent” does not necessarilyhave to mean the same degree of activity, and the activity may beenhanced, or the activity may be decreased as long as there is anactivity higher than that of hA69-KQ/hB26-PF/hAL-AQ according to themeasurement system described above, or preferably F.Xageneration-promoting activity measured under the conditions described in[Example 2] is equivalent to or not less than that ofQ153-G4k/J142-G4h/L180-k.

The above-mentioned antibodies may have one or more amino acidsubstitutions, deletions, additions, and/or insertions in the variableregion (CDR sequences and/or FR sequences) of the amino acid sequence aslong as they have an activity higher than that of hA69-KQ/hB26-PF/hAL-AQaccording to the measurement system described above at page 35, lines11-30, or preferably F.Xa generation-promoting activity measured underthe conditions described in [Example 2] is equivalent to or not lessthan that of Q153-G4k/J142-G4h/L180-k. A method of introducing mutationsinto proteins is well known to those skilled in the art as a method forintroducing one or more amino acid substitutions, deletions, additions,and/or insertions into an amino acid sequence. For example, thoseskilled in the art can prepare a desired mutant functionally equivalentto a multispecific polypeptide multimer having the activity offunctionally substituting for F.VIII by introducing appropriatemutations into the amino acid sequence using site-directed mutagenesis(Hashimoto-Gotoh, T, Mizuno, T, Ogasahara, Y, and Nakagawa, M. (1995) Anoligodeoxyribonucleotide-directed dual amber method for site-directedmutagenesis. Gene 152: 271-275; Zoller, M J, and Smith, M. (1983)Oligonucleotide-directed mutagenesis of DNA fragments cloned into M13vectors. Methods Enzymol. 100: 468-500; Kramer, W, Drutsa, V, Jansen, HW, Kramer, B, Pflugfelder, M, and Fritz, H J (1984) The gapped duplexDNA approach to oligonucleotide-directed mutation construction. NucleicAcids Res. 12, 9441-9456; Kramer W, and Fritz H J (1987)Oligonucleotide-directed construction of mutations via gapped duplex DNAMethods. Enzymol. 154: 350-367; and Kunkel, T A (1985) Rapid andefficient site-specific mutagenesis without phenotypic selection. ProcNatl Acad Sci USA. 82: 488-492) and such.

As such, antibodies of the present invention also include antibodieswith one or more amino acid mutations in the variable region, and havingan activity higher than that of hA69-KQ/hB26-PF/hAL-AQ according to themeasurement system described above at page 35, lines 11-30, orpreferably F.Xa generation-promoting activity measured under theconditions described in [Example 2] is equivalent to or not less thanthat of Q153-G4k/J142-G4h/L180-k.

When an amino acid residue is altered, the amino acid is preferablymutated for a different amino acid(s) that conserves the properties ofthe amino acid side-chain. Examples of amino acid side chain propertiesare: hydrophobic amino acids (A, I, L, M, F, P, W, Y, and V),hydrophilic amino acids (R, D, N, C, E, Q, G, H, K, S, and T), aminoacids containing aliphatic side chains (G, A, V, L, I, and P), aminoacids containing hydroxyl group-containing side chains (S, T, and Y),amino acids containing sulfur-containing side chains (C and M), aminoacids containing carboxylic acid- and amide-containing side chains (D,N, E, and Q), amino acids containing basic side chains (R, K, and H),and amino acids containing aromatic side chains (H, F, Y, and W) (aminoacids are represented by one-letter codes in parentheses) Amino acidsubstitutions within each group are called conservative substitutions.It is already known that a polypeptide containing a modified amino acidsequence in which one or more amino acid residues in a given amino acidsequence are deleted, added, and/or substituted with other amino acidscan retain the original biological activity (Mark, D. F. et al., Proc.Natl. Acad. Sci. USA; (1984) 81: 5662-6; Zoller, M. J. and Smith, M.,Nucleic Acids Res. (1982) 10: 6487-500; Wang, A. et al., Science (1984)224: 1431-3; Dalbadie-McFarland, G. et al., Proc. Natl. Acad. Sci. USA(1982) 79: 6409-13). Such mutants have an amino acid identity of atleast 70%, more preferably at least 75%, even more preferably at least80%, still more preferably at least 85%, yet more preferably at least90%, and most preferably at least 95%, with the variable regions (forexample, CDR sequences, FR sequences, or whole variable regions) of thepresent invention. Herein, sequence identity is defined as thepercentage of residues identical to those in the original amino acidsequence of the heavy chain variable region or light chain variableregion, determined after the sequences are aligned and gaps areappropriately introduced to maximize the sequence identity as necessary.The identity of amino acid sequences can be determined by the methoddescribed below.

Alternatively, the amino acid sequences of variable regions that have asubstitution, deletion, addition, and/or insertion of one or more aminoacids in the amino acid sequence of the variable regions (CDR sequencesand/or FR sequences) and have an activity higher than that ofhA69-KQ/hB26-PF/hAL-AQ according to the measurement system describedabove at page 35, lines 11-30, or preferably F.Xa generation-promotingactivity measured under the conditions described in [Example 2] isequivalent to or not less than that of Q153-G4k/J142-G4h/L180-k can beobtained from nucleic acids that hybridize under stringent conditions tonucleic acid composed of the nucleotide sequence encoding the amino acidsequence of the variable regions. Stringent hybridization conditions toisolate a nucleic acid that hybridizes under stringent conditions to anucleic acid that includes the nucleotide sequence encoding the aminoacid sequence of the variable regions include, for example, theconditions of 6 M urea, 0.4% SDS, 0.5×SSC, and 37° C., or hybridizationconditions with stringencies equivalent thereto. With more stringentconditions, for example, the conditions of 6 M urea, 0.4% SDS, 0.1×SSC,and 42° C., isolation of nucleic acids with a much higher homology canbe expected. The sequences of the isolated nucleic acids can bedetermined by the known methods described below. The overall nucleotidesequence homology of the isolated nucleic acid is at least 50% or highersequence identity, preferably 70% or higher, more preferably 90% orhigher (for example, 95%, 96%, 97%, 98%, 99%, or higher).

Nucleic acids that hybridize under stringent conditions to a nucleicacid composed of the nucleotide sequence encoding the amino acidsequence of the variable regions can also be isolated using, instead ofthe above-described methods using hybridization techniques, geneamplification methods such as polymerase chain reaction (PCR) usingprimers synthesized based on the information of nucleotide sequenceencoding the amino acid sequence of the variable regions.

The identity of one nucleotide sequence or amino acid sequence toanother can be determined using the algorithm BLAST, by Karlin andAltschul (Proc. Natl. Acad. Sci. USA (1993) 90: 5873-7). Programs suchas BLASTN and BLASTX were developed based on this algorithm (Altschul etal., J. Mol. Biol. (1990) 215: 403-10). To analyze nucleotide sequencesaccording to BLASTN based on BLAST, the parameters are set, for example,as score=100 and wordlength=12. On the other hand, parameters used forthe analysis of amino acid sequences by BLASTX based on BLAST include,for example, score=50 and wordlength=3. Default parameters for eachprogram are used when using the BLAST and Gapped BLAST programs.Specific techniques for such analyses are known in the art (see thewebsite of the National Center for Biotechnology Information (NCBI),Basic Local Alignment Search Tool (BLAST); http://www.ncbi.nlm.nih.gov).

The present invention also provides antibodies that bind to an epitopeoverlapping with an epitope bound by the antibodies described above.

Whether an antibody recognizes an epitope overlapping with an epitopethat is recognized by another antibody can be confirmed by thecompetition between the two antibodies against the epitope. Competitionbetween the antibodies can be evaluated by competitive binding assaysusing means such as enzyme-linked immunosorbent assay (ELISA),fluorescence energy transfer method (FRET), and fluorometric microvolumeassay technology (FMAT (Registered trademark)). The amount of antibodiesbound to an antigen indirectly correlate with the binding ability ofcandidate competitor antibodies (test antibodies) that competitivelybind to the overlapping epitope. In other words, as the amount of or theaffinity of test antibodies against the overlapping epitope increases,the amount of antibodies bound to the antigen decreases, and the amountof test antibodies bound to the antigen increases. Specifically,appropriately labeled antibodies and antibodies to be evaluated aresimultaneously added to the antigens, and the thus bound antibodies aredetected using the label. The amount of antibodies bound to the antigencan be easily determined by labeling the antibodies beforehand. Thislabel is not particularly limited, and the labeling method is selectedaccording to the assay technique used. The labeling method includesfluorescent labeling, radiolabeling, enzymatic labeling, and such.

For example, fluorescently labeled antibodies and unlabeled antibodiesor test antibodies are simultaneously added to beads immobilized withF.IX, F.IXa or F.X, and the labeled antibodies are detected byfluorometric microvolume assay technology.

Herein, the “antibody that binds to the overlapping epitope” refers toan antibody that can reduce the binding of the labeled antibody by atleast 50% at a concentration that is usually 100 times higher,preferably 80 times higher, more preferably 50 times higher, even morepreferably 30 times higher, and still more preferably 10 times higherthan a concentration at which the non-labeled antibody reduces thebinding of the labeled antibody by 50% (IC₅₀).

Multispecific antigen-binding molecules, which have antigen-bindingsites of antibodies that bind to epitopes overlapping with epitopesbound by the above-mentioned antibodies, may yield an excellent activityof functionally substituting for F.VIII. Furthermore, in antigen-bindingsites of antibodies that bind to epitopes overlapping with epitopesbound by the above-mentioned antibodies, one or more amino acids may bealtered to obtain a better activity of functionally substituting forF.VIII. Multispecific antigen-binding molecules having a better activityof functionally substituting for F.VIII can be obtained by altering theamino acids of the antigen-binding sites and selecting multispecificantigen-binding molecules having an activity higher than that ofhA69-KQ/hB26-PF/hAL-AQ according to the measurement system describedabove, or preferably having an F.Xa generation-promoting activitymeasured under the conditions described in [Example 2] that isequivalent to or not less than that of Q153-G4k/J142-G4h/L180-k. Toobtain an excellent activity of functionally substituting for F.VIII ofthe present invention, the following amino acid alterations areparticularly preferred.

(1) At least one amino acid residue selected from the amino acidresidues at positions 34, 35, 49, 61, 62, 96, 98, 100, 100b, and 102 byKabat numbering in the H chain of the antibody that recognizes F.IXand/or F.IXa is substituted with a different amino acid.(2) At least one amino acid residue selected from the amino acidresidues at positions 35, 53, 73, 76, 96, 98, 100, and 100a by Kabatnumbering in the H chain of the antibody that recognizes F.X issubstituted with a different amino acid.(3) At least one amino acid residue selected from the amino acidresidues at positions 27, 30, 31, 32, 50, 52, 53, 54, 55, 92, 93, 94,and 95 by Kabat numbering in the antibody L chain is substituted with adifferent amino acid.

Furthermore, in the present invention, preferred antibody amino acidsfor obtaining a better activity of functionally substituting for F.VIIIinclude those mentioned in (4) to (6) below. Regarding these aminoacids, the antibody H chain may originally have such amino acids, orantibody H chain amino acids may be modified to have such a sequence.

(4) An antibody H chain which recognizes F.IX and/or F.IXa, wherein, byKabat numbering, the amino acid residue at position 34 is isoleucine,the amino acid residue at position 35 is asparagine, glutamine, orserine, the amino acid residue at position 49 is serine, the amino acidresidue at position 61 is arginine, the amino acid residue at position62 is glutamic acid, the amino acid residue at position 96 is serine orthreonine, the amino acid residue at position 98 is lysine or arginine,the amino acid residue at position 100 is phenylalanine or tyrosine, theamino acid residue at position 100b is glycine, or the amino acidresidue at position 102 is tyrosine.(5) An antibody H chain which recognizes F.X, wherein, by Kabatnumbering, the amino acid residue at position 35 is aspartic acid, theamino acid residue at position 53 is arginine, the amino acid residue atposition 73 is lysine, the amino acid residue at position 76 is glycine,the amino acid residue at position 96 is lysine or arginine, the aminoacid residue at position 98 is tyrosine, the amino acid residue atposition 100 is tyrosine, or the amino acid residue at position 100a ishistidine.(6) An antibody L chain, wherein, by Kabat numbering, the amino acidresidue at position 27 is lysine or arginine, the amino acid residue atposition 30 is glutamic acid, the amino acid residue at position 31 isarginine, the amino acid residue at position 32 is glutamine, the aminoacid residue at position 50 is arginine or glutamine, the amino acidresidue at position 52 is serine, the amino acid residue at position 53is arginine, the amino acid residue at position 54 is lysine, the aminoacid residue at position 55 is glutamic acid, the amino acid residue atposition 92 is serine, the amino acid residue at position 93 is serine,the amino acid residue at position 94 is proline, or the amino acidresidue at position 95 is proline.

Among the above-mentioned antibody amino acid residues of (1) to (6),favorable positions of amino acid residues for obtaining a particularlyexcellent F.VIII-like activity are shown in the following (1) to (3).

(1) Amino acid residues at positions 34, 35, 61, 98, 100, and 100b,particularly amino acid residues at positions 61 and 100, by Kabatnumbering in the H chain of the antibody that recognizes F.IX and/orF.IXa.(2) Amino acid residues at positions 35, 53, 73, 96, 98, 100, and 100aby Kabat numbering in the H chain of the antibody that recognizes F.X.(3) Amino acid residues at positions 27, 30, 31, 32, 50, 52, 53, 93, 94,and 95, and particularly amino acid residues at positions 27, 30, 31,50, 53, 94, and 95, by Kabat numbering in the antibody L chain.

Specifically, the present invention provides multispecificantigen-binding molecules, wherein a first polypeptide comprises any ofthe antibody H chains selected from the following (a1) to (a14) and anyof the antibody L chains selected from the following (c1) to (c10), andthe second polypeptide comprises any of the antibody H chains selectedfrom the following (b1) to (b12) and any of the antibody L chainsselected from the following (c1) to (c10):

-   -   (a1) an antibody H chain consisting of the amino acid sequence        of SEQ ID NO: 1 (Q1-G4k);    -   (a2) an antibody H chain consisting of the amino acid sequence        of SEQ ID NO: 2 (Q31-z7);    -   (a3) an antibody H chain consisting of the amino acid sequence        of SEQ ID NO: 3 (Q64-z55);    -   (a4) an antibody H chain consisting of the amino acid sequence        of SEQ ID NO: 10 (Q64-z7);    -   (a5) an antibody H chain consisting of the amino acid sequence        of SEQ ID NO: 11 (Q85-G4k);    -   (a6) an antibody H chain consisting of the amino acid sequence        of SEQ ID NO: 12 (Q153-G4k);    -   (a7) an antibody H chain consisting of the amino acid sequence        of SEQ ID NO: 13 (Q354-z106);    -   (a8) an antibody H chain consisting of the amino acid sequence        of SEQ ID NO: 14 (Q360-G4k);    -   (a9) an antibody H chain consisting of the amino acid sequence        of SEQ ID NO: 15 (Q360-z118);    -   (a10) an antibody H chain consisting of the amino acid sequence        of SEQ ID NO: 16 (Q405-G4k);    -   (a11) an antibody H chain consisting of the amino acid sequence        of SEQ ID NO: 17 (Q458-z106);    -   (a12) an antibody H chain consisting of the amino acid sequence        of SEQ ID NO: 18 (Q460-z121);    -   (a13) an antibody H chain consisting of the amino acid sequence        of SEQ ID NO: 19 (Q499-z118);    -   (a14) an antibody H chain consisting of the amino acid sequence        of SEQ ID NO: 20 (Q499-z121);    -   (b1) an antibody H chain consisting of the amino acid sequence        of SEQ ID NO: 4 (J268-G4h);    -   (b2) an antibody H chain consisting of the amino acid sequence        of SEQ ID NO: 5 (J321-G4h);    -   (b3) an antibody H chain consisting of the amino acid sequence        of SEQ ID NO: 6 (J326-z107);    -   (b4) an antibody H chain consisting of the amino acid sequence        of SEQ ID NO: 7 (J344-z107);    -   (b5) an antibody H chain consisting of the amino acid sequence        of SEQ ID NO: 21 (J232-G4h);    -   (b6) an antibody H chain consisting of the amino acid sequence        of SEQ ID NO: 22 (J259-z107);    -   (b7) an antibody H chain consisting of the amino acid sequence        of SEQ ID NO: 23 (J300-z107);    -   (b8) an antibody H chain consisting of the amino acid sequence        of SEQ ID NO: 24 (J327-z107);    -   (b9) an antibody H chain consisting of the amino acid sequence        of SEQ ID NO: 25 (J327-z119);    -   (b10) an antibody H chain consisting of the amino acid sequence        of SEQ ID NO: 26 (J339-z119);    -   (b11) an antibody H chain consisting of the amino acid sequence        of SEQ ID NO: 27 (J346-z107);    -   (b12) an antibody H chain consisting of the amino acid sequence        of SEQ ID NO: 170 (J142-G4h);    -   (c1) an antibody L chain consisting of the amino acid sequence        of SEQ ID NO: 8 (L2-k);    -   (c2) an antibody L chain consisting of the amino acid sequence        of SEQ ID NO: 9 (L45-k);    -   (c3) an antibody L chain consisting of the amino acid sequence        of SEQ ID NO: 28 (L248-k);    -   (c4) an antibody L chain consisting of the amino acid sequence        of SEQ ID NO: 29 (L324-k);    -   (c5) an antibody L chain consisting of the amino acid sequence        of SEQ ID NO: 30 (L334-k);    -   (c6) an antibody L chain consisting of the amino acid sequence        of SEQ ID NO: 31 (L377-k);    -   (c7) an antibody L chain consisting of the amino acid sequence        of SEQ ID NO: 32 (L404-k);    -   (c8) an antibody L chain consisting of the amino acid sequence        of SEQ ID NO: 33 (L406-k);    -   (c9) an antibody L chain consisting of the amino acid sequence        of SEQ ID NO: 34 (L408-k); and    -   (c10) an antibody L chain consisting of the amino acid sequence        of SEQ ID NO: 171 (L180-k).

The present invention also provides multispecific antigen-bindingmolecules, wherein the first polypeptide comprises an antigen-bindingsite which binds to an epitope overlapping with an epitope that binds toan antibody consisting of the antibody H chain of any one of (a1) to(a14) and the antibody L chain of any one of (c1) to (c10) describedabove, and the second polypeptide comprises an antigen-binding sitewhich binds to an epitope overlapping with an epitope that binds to anantibody consisting of the antibody H chain of any one of (b1) to (b12)and the antibody L chain of any one of (c1) to (c10) described above.

Furthermore, the present invention provides multispecificantigen-binding molecules, wherein the first polypeptide comprises anyone antibody H chain selected from the following (e1) to (e3), thesecond polypeptide comprises any one antibody H chain selected from thefollowing (f1) to (f3), and the third polypeptide and the fourthpolypeptide comprise any one antibody L chain selected from thefollowing (g1) to (g4):

-   -   (e1) an H chain of an antibody which binds to an epitope        overlapping with an epitope bound by an antibody consisting of        an antibody H chain of any one of (a1) to (a14) and an antibody        L chain of any one of (c1) to (c10) described above;    -   (e2) an antibody H chain, wherein at least one amino acid        residue selected from the amino acid residues at positions 34,        35, 49, 61, 62, 96, 98, 100, 100b, and 102 by Kabat numbering in        any one antibody H chain selected from (e1) described above is        substituted with another amino acid;    -   (e3) an antibody H chain, wherein by Kabat numbering, the amino        acid residue at position 34 is isoleucine, the amino acid        residue at position 35 is asparagine, glutamine, or serine, the        amino acid residue at position 49 is serine, the amino acid        residue at position 61 is arginine, the amino acid residue at        position 62 is glutamic acid, the amino acid residue at position        96 is serine or threonine, the amino acid residue at position 98        is lysine or arginine, the amino acid residue at position 100 is        phenylalanine or tyrosine, the amino acid residue at position        100b is glycine, or the amino acid residue at position 102 is        tyrosine in any antibody H chain selected from (e1) described        above;    -   (f1) an H chain of an antibody which binds to an epitope        overlapping with an epitope bound by an antibody consisting of        an antibody H chain of any of (b1) to (b12) described above and        an antibody L chain of any of (c1) to (c10) described above;    -   (f2) an antibody H chain, wherein at least one amino acid        residue selected from the amino acid residues at positions 35,        53, 73, 76, 96, 98, 100, and 100a by Kabat numbering in any        antibody H chain of (f1) described above is substituted with        another amino acid;    -   (f3) an antibody H chain, wherein by Kabat numbering, the amino        acid residue at position 35 is aspartic acid, the amino acid        residue at position 53 is arginine, the amino acid residue at        position 73 is lysine, the amino acid residue at position 76 is        glycine, the amino acid residue at position 96 is lysine or        arginine, the amino acid residue at position 98 is tyrosine, the        amino acid residue at position 100 is tyrosine, or the amino        acid residue at position 100a is histidine in any one antibody H        chain selected from (f1) described above;    -   (g1) an L chain of an antibody which binds to an epitope        overlapping with an epitope bound by an antibody which consists        of an antibody H chain of any one of (a1) to    -   (a14) and an antibody L chain of any one of (c1) to (c10)        described above;    -   (g2) an L chain of an antibody which binds to an epitope        overlapping with an epitope bound by an antibody which consists        of an antibody H chain of any one of (b1) to (b12) and an        antibody L chain of any one of (c1) to (c10) described above;    -   (g3) an antibody L chain, wherein at least one amino acid        residue selected from the amino acid residues at positions 27,        30, 31, 32, 50, 52, 53, 54, 55, 92, 93, 94, and 95 by Kabat        numbering in the antibody L chain of either (g1) or (g2)        described above is substituted with another amino acid; and    -   (g4) an antibody L chain, wherein by Kabat numbering, the amino        acid residue at position 27 is lysine or arginine, the amino        acid residue at position 30 is glutamic acid, the amino acid        residue at position 31 is arginine, the amino acid residue at        position 32 is glutamine, the amino acid residue at position 50        is arginine or glutamine, the amino acid residue at position 52        is serine, the amino acid residue at position 53 is arginine,        the amino acid residue at position 54 is lysine, the amino acid        residue at position 55 is glutamic acid, the amino acid residue        at position 92 is serine, the amino acid residue at position 93        is serine, the amino acid residue at position 94 is proline, or        the amino acid residue at position 95 is proline in the antibody        L chain of either (g1) or (g2) described above.

Amino acid substitutions can be performed on the antibodies (clones) ofthe present invention to avoid deamidation, methionine oxidation, andsuch, or to structurally stabilize the antibodies.

The method for obtaining multispecific antigen-binding molecules of thepresent invention is not particularly limited, and may be any method.Bispecific antibodies can be generated according to the methodsdescribed in WO 2006/109592, WO 2005/035756, WO 2006/106905, or WO2007/114325, which are known as examples of the method for producing thebispecific antibodies; and then desired antibodies having a cofactorfunction-substituting activity can be selected and obtained.

For example, the bispecific antibody described in any of the following(a) to (u) is provided by the present invention:

-   -   (a) a bispecific antibody (Q1-G4k/J268-G4h/L45-k), wherein the        first polypeptide is an H chain consisting of the amino acid        sequence of SEQ ID NO: 1, the second polypeptide is an H chain        consisting of the amino acid sequence of SEQ ID NO: 4, and the        third polypeptide and the fourth polypeptide are a commonly        shared L chain of SEQ ID NO: 9;    -   (b) a bispecific antibody (Q1-G4k/J321-G4h/L45-k), wherein the        first polypeptide is an H chain consisting of the amino acid        sequence of SEQ ID NO: 1, the second polypeptide is an H chain        consisting of the amino acid sequence of SEQ ID NO: 5, and the        third polypeptide and the fourth polypeptide are a commonly        shared L chain of SEQ ID NO: 9;    -   (c) a bispecific antibody (Q31-z7/J326-z107/L2-k), wherein the        first polypeptide is an H chain consisting of the amino acid        sequence of SEQ ID NO: 2, the second polypeptide is an H chain        consisting of the amino acid sequence of SEQ ID NO: 6, and the        third polypeptide and the fourth polypeptide are a commonly        shared L chain of SEQ ID NO: 8;    -   (d) a bispecific antibody (Q64-z55/J344-z107/L45-k), wherein the        first polypeptide is an H chain consisting of the amino acid        sequence of SEQ ID NO: 3, the second polypeptide is an H chain        consisting of the amino acid sequence of SEQ ID NO: 7, and the        third polypeptide and the fourth polypeptide are a commonly        shared L chain of SEQ ID NO: 9;    -   (e) a bispecific antibody (Q64-z7/J326-z107/L334-k), wherein the        first polypeptide is an H chain consisting of the amino acid        sequence of SEQ ID NO: 10, the second polypeptide is an H chain        consisting of the amino acid sequence of SEQ ID NO: 6, and the        third polypeptide and the fourth polypeptide are a commonly        shared L chain of SEQ ID NO: 30;    -   (f) a bispecific antibody (Q64-z7/J344-z107/L406-k), wherein the        first polypeptide is an H chain consisting of the amino acid        sequence of SEQ ID NO: 10, the second polypeptide is an H chain        consisting of the amino acid sequence of SEQ ID NO: 7, and the        third polypeptide and the fourth polypeptide are a commonly        shared L chain of SEQ ID NO: 33;    -   (g) a bispecific antibody (Q85-G4k/J268-G4h/L406-k), wherein the        first polypeptide is an H chain consisting of the amino acid        sequence of SEQ ID NO: 11, the second polypeptide is an H chain        consisting of the amino acid sequence of SEQ ID NO: 4, and the        third polypeptide and the fourth polypeptide are a commonly        shared L chain of SEQ ID NO: 33;    -   (h) a bispecific antibody (Q85-G4k/J321-G4h/L334-k), wherein the        first polypeptide is an H chain consisting of the amino acid        sequence of SEQ ID NO: 11, the second polypeptide is an H chain        consisting of the amino acid sequence of SEQ ID NO: 5, and the        third polypeptide and the fourth polypeptide are a commonly        shared L chain of SEQ ID NO: 30;    -   (i) a bispecific antibody (Q153-G4k/J232-G4h/L406-k), wherein        the first polypeptide is an H chain consisting of the amino acid        sequence of SEQ ID NO: 12, the second polypeptide is an H chain        consisting of the amino acid sequence of SEQ ID NO: 21, and the        third polypeptide and the fourth polypeptide are a commonly        shared L chain of SEQ ID NO: 33;    -   (j) a bispecific antibody (Q354-z106/J259-z107/L324-k), wherein        the first polypeptide is an H chain consisting of the amino acid        sequence of SEQ ID NO: 13, the second polypeptide is an H chain        consisting of the amino acid sequence of SEQ ID NO: 22, and the        third polypeptide and the fourth polypeptide are a commonly        shared L chain of SEQ ID NO: 29;    -   (k) a bispecific antibody (Q360-G4k/J232-G4h/L406-k), wherein        the first polypeptide is an H chain consisting of the amino acid        sequence of SEQ ID NO: 14, the second polypeptide is an H chain        consisting of the amino acid sequence of SEQ ID NO: 21, and the        third polypeptide and the fourth polypeptide are a commonly        shared L chain of SEQ ID NO: 33;    -   (l) a bispecific antibody (Q360-z118/J300-z107/L334-k), wherein        the first polypeptide is an H chain consisting of the amino acid        sequence of SEQ ID NO: 15, the second polypeptide is an H chain        consisting of the amino acid sequence of SEQ ID NO: 23, and the        third polypeptide and the fourth polypeptide are a commonly        shared L chain of SEQ ID NO: 30;    -   (m) a bispecific antibody (Q405-G4k/J232-G4h/L248-k), wherein        the first polypeptide is an H chain consisting of the amino acid        sequence of SEQ ID NO: 16, the second polypeptide is an H chain        consisting of the amino acid sequence of SEQ ID NO: 21, and the        third polypeptide and the fourth polypeptide are a commonly        shared L chain of SEQ ID NO: 28;    -   (n) a bispecific antibody (Q458-z106/J346-z107/L408-k), wherein        the first polypeptide is an H chain consisting of the amino acid        sequence of SEQ ID NO: 17, the second polypeptide is an H chain        consisting of the amino acid sequence of SEQ ID NO: 27, and the        third polypeptide and the fourth polypeptide are a commonly        shared L chain of SEQ ID NO: 34;    -   (o) a bispecific antibody (Q460-z121/J327-z119/L334-k), wherein        the first polypeptide is an H chain consisting of the amino acid        sequence of SEQ ID NO: 18, the second polypeptide is an H chain        consisting of the amino acid sequence of SEQ ID NO: 25, and the        third polypeptide and the fourth polypeptide are a commonly        shared L chain of SEQ ID NO: 30;    -   (p) a bispecific antibody (Q499-z118/J327-z107/L334-k), wherein        the first polypeptide is an H chain consisting of the amino acid        sequence of SEQ ID NO: 19, the second polypeptide is an H chain        consisting of the amino acid sequence of SEQ ID NO: 24, and the        third polypeptide and the fourth polypeptide are a commonly        shared L chain of SEQ ID NO: 30;    -   (q) a bispecific antibody (Q499-z118/J327-z107/L377-k), wherein        the first polypeptide is an H chain consisting of the amino acid        sequence of SEQ ID NO: 19, the second polypeptide is an H chain        consisting of the amino acid sequence of SEQ ID NO: 24, and the        third polypeptide and the fourth polypeptide are a commonly        shared L chain of SEQ ID NO: 31;    -   (r) a bispecific antibody (Q499-z118/J346-z107/L248-k), wherein        the first polypeptide is an H chain consisting of the amino acid        sequence of SEQ ID NO: 19, the second polypeptide is an H chain        consisting of the amino acid sequence of SEQ ID NO: 27, and the        third polypeptide and the fourth polypeptide are a commonly        shared L chain of SEQ ID NO: 28;    -   (s) a bispecific antibody (Q499-z121/J327-z119/L404-k), wherein        the first polypeptide is an H chain consisting of the amino acid        sequence of SEQ ID NO: 20, the second polypeptide is an H chain        consisting of the amino acid sequence of SEQ ID NO: 25, and the        third polypeptide and the fourth polypeptide are a commonly        shared L chain of SEQ ID NO: 32;    -   (t) a bispecific antibody (Q499-z121/J339-z119/L377-k), wherein        the first polypeptide is an H chain consisting of the amino acid        sequence of SEQ ID NO: 20, the second polypeptide is an H chain        consisting of the amino acid sequence of SEQ ID NO: 26, and the        third polypeptide and the fourth polypeptide are a commonly        shared L chain of SEQ ID NO: 31; and    -   (u) a bispecific antibody (Q153-G4k/J142-G4h/L180-k), wherein        the first polypeptide is an H chain consisting of the amino acid        sequence of SEQ ID NO: 12, the second polypeptide is an H chain        consisting of the amino acid sequence of SEQ ID NO: 170, and the        third polypeptide and the fourth polypeptide are a commonly        shared L chain of SEQ ID NO: 171.

Amino acid sequences, molecular weights, isoelectric points, or presenceor absence and form of sugar chains of the antibodies of the presentinvention vary depending on cells or hosts that produce the antibodiesor purification methods described later. However, as long as theobtained antibodies have functions equivalent to the antibodies of thepresent invention, they are included in the present invention. Forexample, when an antibody of the present invention is expressed inprokaryotic cells such as E. coli, a methionine residue will be added tothe N terminus of the original antibody amino acid sequence. Antibodiesof the present invention also comprise such antibodies.

Bispecific antibodies of the present invention can be produced bymethods known to those skilled in the art.

Based on the obtained sequence of the anti-F.IX/F.IXa antibody oranti-F.X antibody, the anti-F.IX/F.IXa antibody or anti-F.X antibody canbe prepared, for example, by genetic recombination techniques known tothose skilled in the art. Specifically, a polynucleotide encoding anantibody can be constructed based on the sequence of the anti-F.IX/F.IXaantibody or anti-F.X antibody, inserted into an expression vector, andthen expressed in appropriate host cells (see for example, Co, M. S. etal., J. Immunol. (1994) 152, 2968-2976; Better, M. and Horwitz, A. H.,Methods Enzymol. (1989) 178, 476-496; Pluckthun, A. and Skerra, A.,Methods Enzymol. (1989) 178, 497-515; Lamoyi, E., Methods Enzymol.(1986) 121, 652-663; Rousseaux, J. et al., Methods Enzymol. (1986) 121,663-669; Bird, R. E. and Walker, B. W., Trends Biotechnol. (1991) 9,132-137).

The vectors include M13 vectors, pUC vectors, pBR322, pBluescript, andpCR-Script. Alternatively, when aiming to subclone and excise cDNA, thevectors include, for example, pGEM-T, pDIRECT, and pT7, in addition tothe vectors described above. Expression vectors are particularly usefulwhen using vectors for producing the antibodies of the presentinvention. For example, when aiming for expression in E. coli such asJM109, DHSα, HB101, and XL1-Blue, the expression vectors not only havethe characteristics that allow vector amplification in E. coli, but mustalso carry a promoter that allows efficient expression in E. coli, forexample, lacZ promoter (Ward et al., Nature (1989) 341: 544-546; FASEBJ. (1992) δ: 2422-2427), araB promoter (Better et al., Science (1988)240: 1041-1043), T7 promoter or such. Such vectors include pGEX-5X-1(Pharmacia), “QIAexpress system” (Qiagen), pEGFP, or pET (in this case,the host is preferably BL21 that expresses T7 RNA polymerase) inaddition to the vectors described above.

The expression plasmid vectors may contain signal sequences for antibodysecretion. As a signal sequence for antibody secretion, a pelB signalsequence (Lei, S. P. et al J. Bacteriol. (1987) 169: 4379) may be usedwhen a protein is secreted into the E. coli periplasm. The vector can beintroduced into host cells by calcium chloride or electroporationmethods, for example.

In addition to vectors for E. coli, the vectors for producing theantibodies of the present invention include mammalian expression vectors(for example, pcDNA3 (Invitrogen), pEF-BOS (Nucleic Acids. Res. 1990,18(17): p5322), pEF, and pCDM8), insect cell-derived expression vectors(for example, the “Bac-to-BAC baculovirus expression system” (Gibco-BRL)and pBacPAK8), plant-derived expression vectors (for example, pMH1 andpMH2), animal virus-derived expression vectors (for example, pHSV, pMV,and pAdexLcw), retroviral expression vectors (for example, pZIPneo),yeast expression vectors (for example, “Pichia Expression Kit”(Invitrogen), pNV11, and SP-Q01), and Bacillus subtilis expressionvectors (for example, pPL608 and pKTH50), for example.

When aiming for expression in animal cells such as CHO, COS, and NIH3T3cells, the expression plasmid vectors must have a promoter essential forexpression in cells, for example, SV40 promoter (Mulligan et al., Nature(1979) 277: 108), MMLV-LTR promoter, EF1α promoter (Mizushima et al.,Nucleic Acids Res. (1990) 18: 5322), and CMV promoter, and morepreferably they have a gene for selecting transformed cells (forexample, a drug resistance gene that allows evaluation using an agent(neomycin, G418, or such)). Vectors with such characteristics includepMAM, pDR2, pBK-RSV, pBK-CMV, pOPRSV, and pOP13, for example.

In addition, the following method can be used for stable gene expressionand gene amplification in cells: CHO cells deficient in a nucleic acidsynthesis pathway are introduced with a vector that carries a DHFR genewhich compensates for the deficiency (for example, pSV2-dhfr (MolecularCloning 2^(nd) edition, Cold Spring Harbor Laboratory Press, 1989)), andthe vector is amplified using methotrexate (MTX). Alternatively, thefollowing method can be used for transient gene expression: COS cellswith a gene expressing SV40 T antigen on their chromosome aretransformed with a vector with an SV40 replication origin (pcD andsuch). Replication origins derived from polyoma virus, adenovirus,bovine papilloma virus (BPV), and such can also be used. To amplify genecopy number in host cells, the expression vectors may further carryselection markers such as aminoglycoside transferase (APH) gene,thymidine kinase (TK) gene, E. coli xanthine-guaninephosphoribosyltransferase (Ecogpt) gene, and dihydrofolate reductase(dhfr) gene.

The antibodies of the present invention obtained by the methodsdescribed above can be isolated from inside host cells or from outsidethe cells (the medium, or such), and purified to homogeneity. Theantibodies can be isolated and purified by methods routinely used forisolating and purifying antibodies, and the type of method is notlimited. For example, the antibodies can be isolated and purified byappropriately selecting and combining column chromatography, filtration,ultrafiltration, salting-out, solvent precipitation, solvent extraction,distillation, immunoprecipitation, SDS-polyacrylamide gelelectrophoresis, isoelectrofocusing, dialysis, recrystallization, andsuch.

The chromatographies include, for example, affinity chromatography, ionexchange chromatography, hydrophobic chromatography, gel filtration,reverse phase chromatography, and adsorption chromatography (Strategiesfor Protein Purification and Characterization: A Laboratory CourseManual. Ed Daniel R. Marshak et al., Cold Spring Harbor LaboratoryPress, 1996). The chromatographic methods described above can beconducted using liquid chromatography, for example, HPLC and FPLC.Columns that can be used for affinity chromatography include protein Acolumns and protein G columns. Columns using protein A include, forexample, Hyper D, POROS, and Sepharose FF (GE Amersham Biosciences). Thepresent invention includes antibodies that are highly purified usingthese purification methods.

The obtained antibodies can be purified to homogeneity. Separation andpurification of the antibodies can be performed using conventionalseparation and purification methods used for ordinary proteins. Forexample, the antibodies can be separated and purified by appropriatelyselecting and combining column chromatography such as affinitychromatography, filtration, ultrafiltration, salting-out, dialysis, SDSpolyacrylamide gel electrophoresis, isoelectric focusing, and such,without limitation (Antibodies: A Laboratory Manual. Ed Harlow and DavidLane, Cold Spring Harbor Laboratory, 1988). Columns used for affinitychromatography include, for example, protein A columns and protein Gcolumns.

In one embodiment of antibodies of the present invention, since theantibodies of the present invention functionally substitute for cofactorF.VIII, they are expected to become effective pharmaceutical agentsagainst diseases resulting from decrease in activity (function) of thiscofactor. Examples of the above-mentioned diseases include bleeding,diseases accompanying bleeding, or a disease caused by bleeding. Inparticular, there may have excellent therapeutic effects on hemophilias,in which bleeding disorders are caused by a deficiency or decrease ofF.VIII/F.VIIIa function. Among the hemophilias, they are expected tobecome excellent therapeutic agents for hemophilia A, in which bleedingdisorders are caused by a hereditary deficiency or decrease ofF.VIII/F.VIIIa function.

The present invention provides (pharmaceutical) compositions comprisingthe antibodies of the present invention and pharmaceutically acceptablecarriers. For example, the antibodies of the present invention thatrecognize both F.IX or F.IXa and F.X, and functionally substitute forF.VIII are expected to become pharmaceuticals (pharmaceuticalcompositions) or pharmaceutical agents for preventing and/or treatingbleeding, diseases accompanying bleeding, diseases caused by bleeding,and the like.

In the context of the present invention, bleeding, diseases accompanyingbleeding, and/or diseases caused by bleeding preferably refer todiseases that develop and/or progress due to reduction or deficiency inactivity of F.VIII and/or activated coagulation factor VIII (F.VIIIa).Such diseases include the above-described hemophilia A, diseases inwhich an inhibitor against F.VIII/F.VIIIa appear, acquired hemophilia,von Willebrand's disease, and such, but are not particularly limitedthereto.

Pharmaceutical compositions used for therapeutic or preventive purposes,which comprise antibodies of the present invention as activeingredients, can be formulated by mixing, if necessary, with suitablepharmaceutically acceptable carriers, vehicles, and such that areinactive against the antibodies. For example, sterilized water,physiological saline, stabilizers, excipients, antioxidants (such asascorbic acid), buffers (such as phosphate, citrate, histidine, andother organic acids), antiseptics, surfactants (such as PEG and Tween),chelating agents (such as EDTA), and binders may be used. They may alsocomprise other low-molecular-weight polypeptides, proteins such as serumalbumin, gelatin, and immunoglobulins, amino acids such as glycine,glutamine, asparagine, glutamic acid, asparagic acid, methionine,arginine, and lysine, sugars and carbohydrates such as polysaccharidesand monosaccharides, and sugar alcohols such as mannitol and sorbitol.When preparing an aqueous solution for injection, physiological salineand isotonic solutions comprising glucose and other adjuvants such asD-sorbitol, D-mannose, D-mannitol, and sodium chloride may be used, andif necessary, in combination with appropriate solubilizers such asalcohol (for example, ethanol), polyalcohols (such as propylene glycoland PEG), and nonionic surfactants (such as polysorbate 80, polysorbate20, poloxamer 188, and HCO-50). By mixing hyaluronidase into theformulation, a larger fluid volume can be administered subcutaneously(Expert Opin Drug Deliv. 2007 July; 4(4): 427-40).

If necessary, antibodies of the present invention may be encapsulated inmicrocapsules (e.g., those made of hydroxymethylcellulose, gelatin, andpoly(methylmetacrylate)), or incorporated as components of colloidaldrug delivery systems (e.g., liposomes, albumin microspheres,microemulsion, nanoparticles, and nanocapsules) (see, for example,“Remington's Pharmaceutical Science 16th edition”, Oslo Ed. (1980)).Methods for preparing the pharmaceutical agents as controlled-releasepharmaceutical agents are also well known, and such methods may beapplied to the antibodies of the present invention (Langer et al., J.Biomed. Mater. Res. 15: 267-277 (1981); Langer, Chemtech. 12: 98-105(1982); U.S. Pat. No. 3,773,919; European Patent Application PublicationNo. EP 58,481; Sidman et al., Biopolymers 22: 547-556 (1983); EP133,988).

The dose of a pharmaceutical composition of the present invention may beappropriately determined by considering the dosage form, method ofadministration, patient age and body weight, symptoms of the patient,type of the disease, and degree of progress of the disease, and isultimately decided by physicians. Generally, the daily dose for an adultis 0.1 mg to 2,000 mg at once or in several portions. The dose is morepreferably 0.2 to 1,000 mg/day, even more preferably 0.5 to 500 mg/day,still more preferably 1 to 300 mg/day, yet more preferably 3 to 100mg/day, and most preferably 5 to 50 mg/day. These doses may vary,depending on the patient body weight and age, and the method ofadministration; however, selection of suitable dosage is well within thepurview of those skilled in the art. Similarly, the dosing period may beappropriately determined depending on the therapeutic progress.

Furthermore, the present invention provides genes or nucleic acidsencoding the antibodies of the present invention. In addition, genetherapy may be performed by incorporating genes or nucleic acidsencoding the antibodies of the present invention into vectors for genetherapy. In addition to direct administration using naked plasmids,methods of administration include administration after packaging intoliposomes and such, forming a variety of virus vectors such asretrovirus vectors, adenovirus vectors, vaccinia virus vectors, poxvirusvectors, adeno-associated virus vectors, and HVJ vectors (see Adolph“Viral Genome Methods” CRC Press, Florida (1996)), or coating withcarrier beads such as colloidal gold particles (WO 93/17706, and such).However, so long as the antibodies are expressed in vivo and theiractivities are exercised, any method can be used for administration.Preferably, a sufficient dose can be administered by a suitableparenteral route (such as injecting or infusing intravenously,intraperitoneally, subcutaneously, intradermally, intramuscularly, intoadipose tissues or mammary glands; inhalation; gas-driven particlebombardment (using electron gun and such); or mucosal route such asnasal drops). Alternatively, the genes encoding the antibodies of thepresent invention may be administered into blood cells, bone marrowcells, and such ex vivo using liposome transfection, particlebombardment (U.S. Pat. No. 4,945,050), or viral infection, and the cellsmay be reintroduced into patients. Any gene encoding an antibody of thepresent invention may be used in gene therapy, and its examples includegenes comprising nucleotide sequences encoding the CDRs of Q1, Q31, Q64,Q85, Q153, Q354, Q360, Q405, Q458, Q460, Q499, J232, J259, J268, J300,J321, J326, J327, J339, J344, J346, J142, L2, L45, L248, L324, L334,L377, L404, L406, L408, and L180 described above.

The present invention also provides methods for preventing and/ortreating bleeding, diseases accompanying bleeding, and/or diseasescaused by bleeding, such methods comprising the step of administeringthe antibodies or compositions of the present invention. The antibodiesor compositions can be administered, for example, by the above-mentionedmethods.

Furthermore, the present invention provides kits to be used for theabove-mentioned methods, such kits comprising at least an antibody orcomposition of the present invention. In addition, the kits may include,packaged therewith, a syringe, injection needle, pharmaceuticallyacceptable medium, alcohol-soaked cotton, adhesive bandage, instructionsdescribing the method of use, and the like.

The present invention also relates to use of a multispecificantigen-binding molecule, a bispecific antibody, or a composition of thepresent invention in the manufacture of an agent for preventing and/ortreating bleeding, a disease accompanying bleeding, or a disease causedby bleeding.

Furthermore, the present invention relates to a multispecificantigen-binding molecule, a bispecific antibody, or a composition of thepresent invention for preventing and/or treating bleeding, a diseaseaccompanying bleeding, or a disease caused by bleeding.

All prior art references cited herein are incorporated by reference intothis description.

EXAMPLES

Herein below, the present invention will be specifically described withreference to the Examples, but it is not to be construed as beinglimited thereto.

Example 1 Production of Bispecific Antibodies Having F.XaGeneration-Promoting Activity

In WO 2006/109592, hA69-KQ/hB26-PF/hAL-AQ was obtained as a bispecificantibody having an activity of functionally substituting for F.VIII.However, there was the possibility that this antibody has an inhibitingaction on the reaction in which F.IXa activates F.X using F.VIIIa as acofactor.

As shown in FIG. 1, antibodies that bind to F.IX/F.IXa or F.X mayinhibit the formation of the F.IXa-F.VIIIa complex (Factor Xase(F.Xase)), or inhibit F.Xase activity (activation of F.X). Hereafter,inhibition of F.Xase formation and/or action of inhibiting F.Xaseactivity will be mentioned as F.Xase inhibitory action. F.Xaseinhibitory action is the inhibition of a coagulation reaction in whichF.VIIIa serves as the cofactor, which may suppress the remaining F.VIIIfunction in a patient or the function of the administered F.VIIIformulation. Therefore, it is desirable that F.Xa generation-promotingactivity, which is the objective of the bispecific antibody, is high,while F.Xase inhibitory action is low. In particular, for patientsmaintaining F.VIII function and patients receiving treatment with aF.VIII formulation, it is more desirable that F.Xa generation-promotingactivity and F.Xase inhibitory action are separated as much as possible.

However, F.Xase inhibitory action is due to the binding to the antigen(F.IXa and/or F.X), which is fundamental property of the antibody. Onthe other hand, a bispecific antibody having F.Xa generation-promotingaction (functionally substituting for F.VIII) also needs to bind to theantigens (F.IXa and F.X). Therefore, it is predicted that it isextremely difficult to obtain bispecific antibodies that do not have anF.Xase inhibitory action but have an F.Xa generation-promoting activity(functionally substituting for F.VIII). Similarly, it is predicted thatit is extremely difficult to decrease an F.Xase inhibitory action whileincreasing the target F.Xa generation-promoting activity by introducingamino acid substitutions in a bispecific antibody.

The present inventors prepared genes for approximately 200 types ofantibodies against human F.IXa and human F.X, respectively, using amethod known to those skilled in the art, which is the method ofobtaining antibody genes from antibody-producing cells of animalsimmunized with an antigen (human F.IXa or human F.X), and introducingamino acid substitutions, when necessary. Each antibody gene wasincorporated into an animal cell expression vector.

40,000 or more bispecific antibodies as anti-F.IXa antibody and anti-F.Xantibody combinations were transiently expressed by simultaneouslytransfecting the anti-human F.IXa antibody H chain expression vector,the anti-human F.X antibody H chain expression vector, and the commonlyshared antibody L chain expression vector into mammalian cells such asHEK293H cells. As a comparative control, bispecific antibodyhA69-KQ/hB26-PF/hAL-AQ (SEQ ID NOs: 165/166/167) described in WO2006/109592 was prepared.

Since the mutations mentioned in WO 2006/106905 or WO 1996/027011 wereintroduced into the CH3 domain of each H chain, it was thought thatbispecific antibodies were mainly expressed. Antibodies in the cellculture supernatant were purified by a method known to those skilled inthe art using Protein A.

The present inventors measured the F.Xa generation-promoting activity ofthese antibodies by the method described below. All reactions wereperformed at room temperature.

Five μL of antibody solution diluted with Tris-buffered salinecontaining 0.1% bovine serum albumin (hereafter referred to as TBSB) wasmixed with 2.5 μL of 27 ng/mL Human Factor IXa beta (Enzyme ResearchLaboratories) and 2.5 μL of 6 IU/mL of Novact (registered trademark) M(Kaketsuken), and then incubated in a 384-well plate at room temperaturefor 30 minutes.

The enzyme reaction in this mixed solution was initiated by adding 5 μLof 24.7 μg/mL of Human Factor X (Enzyme Research Laboratories), and tenminutes later, 5 μL of 0.5 M EDTA was added to stop the reaction. Thecoloring reaction was initiated by adding 5 μL of coloring substratesolution. After a 50-minute coloring reaction, the change in absorbanceat 405 nm was measured using the SpectraMax 340PC³⁸⁴ (MolecularDevices). F.Xa generation-promoting activity was indicated as the valueobtained by subtracting the absorbance of the antibody-free reactionsolution from the absorbance of the antibody-supplemented reactionsolution.

TBCP (TBSB containing 93.75 μM phospholipid solution (SYSMEX CO.), 7.5mM CaCl₂, and 1.5 mM MgCl₂) was used as the solvent for Human FactorIXa, Novact (registered trademark) M, and Human Factor X. A coloringsubstrate solution S-2222™ (CHROMOGENIX) was dissolved in purified waterat 1.47 mg/mL, and then used in this assay.

To evaluate the F.Xase inhibitory action of the antibodies, the presentinventors measured the effects on F.X activation by F.IXa in thepresence of F.VIIIa using the following method. All reactions wereperformed at room temperature.

Five μL of antibody solution diluted with Tris-buffered salinecontaining 0.1% bovine serum albumin (hereafter referred to as TBSB) wasmixed with 2.5 μL of 80.9 ng/mL Human Factor IXa beta (Enzyme ResearchLaboratories), and then incubated in a 384-well plate at roomtemperature for 30 minutes.

2.5 μL of 1.8 IU/mL of F.VIIIa (production method will be descriedlater) was further added, and 30 seconds later, the enzyme reaction inthis mixed solution was initiated by adding 5 μL of 24.7 μg/mL of HumanFactor X (Enzyme Research Laboratories). Six minutes later, 5 μL of 0.5M EDTA was added to stop the reaction. The coloring reaction wasinitiated by adding 5 μL of coloring substrate solution. After a14-minute coloring reaction, the change in absorbance at 405 nm wasmeasured using the SpectraMax 340PC³⁸⁴ (Molecular Devices). F.Xaseinhibitory action of an antibody was indicated as the value obtained bysubtracting the absorbance of the antibody-free reaction solution fromthe absorbance of the antibody-supplemented reaction solution.

F.VIIIa was prepared by mixing 5.4 IU/mL of Kogenate (registeredtrademark) FS (Bayer HealthCare) and 1.11 μg/mL of Human alpha Thrombin(Enzyme Research Laboratories) at a volume ratio of 1:1, incubating atroom temperature for one minute, and then adding 7.5 U/mL of Hirudin(Merck KgaA) at a quantity that is half the volume of the mixturesolution. The prepared solution was defined as 1.8 IU/mL of FVIIIa, andone minute after addition of Hirudin, this was used for assays.

TBCP (TBSB containing 93.75 μM phospholipid solution (SYSMEX CO.), 7.5mM CaCl₂, and 1.5 mM MgCl₂) was used for the solvent for Human FactorIXa, Human Factor X, Kogenate (registered trademark) FS, Human alphaThrombin, and Hirudin. A coloring substrate solution S-2222™(CHROMOGENIX) was dissolved in purified water at 1.47 mg/mL, and thenused in this assay.

The F.Xa generation-promoting activities of each of the bispecificantibodies are indicated in FIGS. 3 and 4, and the F.Xase inhibitoryactions of each of the bispecific antibodies are indicated in FIG. 5.Various amino acid substitutions that increase the F.Xageneration-promoting activity have been found, but as expected, most ofthe amino acid substitutions that increase the F.Xa generation-promotingactivity increased F.Xase inhibitory action as well, and suppressingF.Xase inhibitory action while increasing F.Xa generation-promotingactivity was very difficult.

Under such circumstances, the inventors of the present applicationobtained Q1-G4k/J268-G4h/L45-k, Q1-G4k/J321-G4h/L45-k,Q31-z7/J326-z107/L2-k, Q64-z55/J344-z107/L45-k as bispecific antibodieswith a high F.Xa generation-promoting activity and a low F.Xaseinhibitory action. In addition, Q1-G4k (SEQ ID NO: 1), Q31-z7 (SEQ IDNO: 2), and Q64-z55 (SEQ ID NO: 3) were obtained as anti-human F.IXaantibody H chains, J268-G4h (SEQ ID NO: 4), J321-G4h (SEQ ID NO: 5),J326-z107 (SEQ ID NO: 6), and J344-z107 (SEQ ID NO: 7) were obtained asprototype anti-human F.X antibody H chains, and L2-k (SEQ ID NO: 8) andL45-k (SEQ ID NO: 9) were obtained as prototype commonly shared antibodyL chains. The character before the hyphen in the sequence name indicatesthe variable region and the character after the hyphen indicates theconstant region. Each bispecific antibody name is indicated by listingthe sequence names of each chain to be transfected.

Most of the bispecific antibodies having F.Xa generation-promotingactivity close to that of hA69-KQ/hB26-PF/hAL-AQ had high F.Xaseinhibitory action as expected, but these bispecific antibodies(Q1-G4k/J268-G4h/L45-k, Q1-G4k/J321-G4h/L45-k, Q31-z7/J326-z107/L2-k,Q64-z55/J344-z107/L45-k) were found to have higher F.Xageneration-promoting activity and lower F.Xase inhibitory action thanhA69-KQ/hB26-PF/hAL-AQ described in WO 2006/109592. The presentinventors conducted examinations to further increase the F.Xageneration-promoting activity and reduce the F.Xase inhibitory actionusing these four antibodies as prototype antibodies. Screening ofbispecific antibodies that increase F.Xa generation-promoting activityand reduce F.Xase inhibitory action is indicated in FIG. 2.

Example 2 Production of Modified Antibodies

The present inventors introduced various combinations of amino acidmutations that affect the F.Xa generation-promoting activities andF.Xase inhibitory actions found in Example 1 to each of the chains ofthe prototype antibodies by a method known to those skilled in the artsuch as PCR for introducing mutations and evaluated the combinations ofmodified chains on a large scale to screen for amino acid substitutionsthat will further increase the F.Xa generation-promoting activities andreduce the F.Xase inhibitory actions of the four prototype antibodies.

Each of the modified bispecific antibodies with amino acid substitutionswere expressed transiently and purified by methods similar to those forthe prototype antibodies. The F.Xa generation-promoting activities ofthe antibodies were measured using the following method. All reactionswere performed at room temperature.

Five μL of antibody solution diluted with Tris-buffered salinecontaining 0.1% bovine serum albumin (hereafter referred to as TBSB) wasmixed with 2.5 μL of 27 ng/mL Human Factor IXa beta (Enzyme ResearchLaboratories) and 2.5 μL of 6 IU/mL of Novact (registered trademark) M(Kaketsuken), and then incubated in a 384-well plate at room temperaturefor 30 minutes.

The enzyme reaction in this mixed solution was initiated by adding 5 μLof 24.7 μg/mL of Human Factor X (Enzyme Research Laboratories), and twominutes later, 5 μL of 0.5 M EDTA was added to stop the reaction. Thecoloring reaction was initiated by adding 5 μL of coloring substratesolution. After a 20-minute coloring reaction, the change in absorbanceat 405 nm was measured using the SpectraMax 340PC³⁸⁴ (MolecularDevices). F.Xa generation-promoting activity was indicated as the valueobtained by subtracting the absorbance of the antibody-free reactionsolution from the absorbance of the antibody-supplemented reactionsolution.

TBCP (TBSB containing 93.75 μM phospholipid solution (SYSMEX CO.), 7.5mM CaCl₂, and 1.5 mM MgCl₂) was used as the solvent for Human FactorIXa, Novact (registered trademark) M, and Human Factor X. A coloringsubstrate solution S-2222™ (CHROMOGENIX) was dissolved in purified waterat 1.47 mg/mL, and then used in this assay.

F.Xase inhibitory actions of the antibodies were also evaluated bypreviously described methods.

The F.Xa generation-promoting activities of each of the modifiedbispecific antibodies are indicated in FIG. 4, and the F.Xase inhibitoryactions of each of the bispecific antibodies are indicated in FIG. 5.

The inventors of the present application obtainedQ85-G4k/J268-G4h/L406-k, Q85-G4k/J321-G4h/L334-k,Q64-z7/J344-z107/L406-k, and Q64-z7/J326-z107/L334-k as bispecificantibodies with a high F.Xa generation-promoting activity and a lowF.Xase inhibitory action. In addition, they discovered Q64-z7 (SEQ IDNO: 10) and Q85-G4k (SEQ ID NO: 11) as the anti-human F.IXa antibody Hchain, and L334-k (SEQ ID NO: 30) and L406-k (SEQ ID NO: 33) as thecommonly shared antibody L chains with increased F.Xageneration-promoting activity. Though F.Xase inhibitory actionsincreased slightly, F.Xa generation-promoting activity increased greatlyin Q85-G4k/J268-G4h/L406-k, Q85-G4k/J321-G4h/L334-k,Q64-z7/J344-z107/L406-k, and Q64-z7/J326-z107/L334-k. Since thesemodified antibodies have very large F.Xa generation-promoting activitiescompared to increase in F.Xase inhibitory actions, the F.Xageneration-promoting activity and the F.Xase inhibitory action couldfurther be separated compared to the prototype antibodies. This way,combinations that suppress the F.Xase inhibitory action and increase theF.Xa generation-promoting activity were discovered.

While a higher F.Xa generation-promoting activity is preferred for thediscovered prototype antibodies to functionally substitute for F.VIII bybispecific antibodies, lower F.Xase inhibitory action was consideredfavorable to clinically use for patients maintaining F.VIII functions orpatients receiving treatment with F.VIII formulations. Therefore,further modifications were performed to produce bispecific antibodies inwhich F.Xase inhibitory action is not increased while F.Xageneration-promoting activity is further increased.

As a result, Q153-G4k/J232-G4h/L406-k, Q354-z106/J259-z107/L324-k,Q360-G4k/J232-G4h/L406-k, Q360-z118/J300-z107/L334-k,Q405-G4k/J232-G4h/L248-k, Q458-z106/J346-z107/L408-k,Q460-z121/J327-z119/L334-k, Q499-z118/J327-z107/L334-k,Q499-z118/J327-z107/L377-k, Q499-z118/J346-z107/L248-k,Q499-z121/J327-z119/L404-k, Q499-z121/J339-z119/L377-k, andQ153-G4k/J142-G4h/L180-k were obtained as bispecific antibodies with ahigh F.Xa generation-promoting activity and a low F.Xase inhibitoryaction. In addition, the Inventors discovered Q153-G4k (SEQ ID NO: 12),Q354-z106 (SEQ ID NO: 13), Q360-G4k (SEQ ID NO: 14), Q360-z118 (SEQ IDNO: 15), Q405-G4k (SEQ ID NO: 16), Q458-z106 (SEQ ID NO: 17), Q460-z121(SEQ ID NO: 18), Q499-z118 (SEQ ID NO: 19), and Q499-z121 (SEQ ID NO:20) as the anti-human F.IXa antibody H chain, J232-G4h (SEQ ID NO: 21),J259-z107

(SEQ ID NO: 22), J300-z107 (SEQ ID NO: 23), J327-z107 (SEQ ID NO: 24),J327-z119 (SEQ ID NO: 25), J339-z119 (SEQ ID NO: 26), J346-z107 (SEQ IDNO: 27), J142-G4h (SEQ ID NO: 170) as the anti-human F.X antibody Hchains with increased F.Xa generation-promoting activity, and L248-k(SEQ ID NO: 28), L324-k (SEQ ID NO: 29), L377-k (SEQ ID NO: 31),

L404-k (SEQ ID NO: 32), L408-k (SEQ ID NO: 34), and L180-k (SEQ ID NO:171) as the commonly shared antibody L chains.

Since these antibodies have very high F.Xa generation-promotingactivities while having suppressed F.Xase inhibitory actions, they mayhave very useful properties for patients maintaining an F.VIII functionand patients receiving treatment with F.VIII formulations. Sinceantibodies generally have long half-lives, and can be administeredsubcutaneously, these bispecific antibodies may be of great value tohemophilia A patients, when compared to existing replacement therapy byintravenous administration of existing F.VIII formulations forhemophilia A.

Sequence comparisons of the variable regions of each of the chains usedin Example 1 and Example 2 are shown in FIGS. 6A to D. For example, toenhance the F.Xa generation-promoting activity of a bispecific antibody,the following amino acids were found to be important: in the anti-humanF.IXa antibody H chain, isoleucine at position 34, asparagine,glutamine, or serine at position 35, serine at position 49, arginine atposition 61, glutamic acid at position 62, serine or threonine atposition 96, lysine or arginine at position 98, serine or glutamic acidat position 99, phenylalanine or tyrosine at position 100, glycine atposition 100b, tyrosine at position 102, and such; in the anti-human F.Xantibody H chain, aspartic acid at position 35, arginine at position 53,lysine at position 73, glycine at position 76, lysine or arginine atposition 96, tyrosine at position 98, tyrosine at position 100,histidine at position 100a, and such; in the commonly shared antibody Lchain, lysine or arginine at position 27, glutamic acid at position 30,arginine at position 31, glutamine at position 32, arginine or glutamineat position 50, serine at position 52, arginine at position 53, lysineat position 54, glutamic acid at position 55, serine at position 92,serine at position 93, proline at position 94, proline at position 95,and such (the variable region amino acids are numbered by Kabatnumbering (Kabat E A et al. 1991. Sequences of Proteins of ImmunologicalInterest. NIH)).

INDUSTRIAL APPLICABILITY

The present invention provides multispecific antigen-binding moleculeshaving a high activity of functionally substituting for F.VIII, whichare antibodies that recognize both an enzyme and its substrate.Furthermore, the present invention provides multispecificantigen-binding molecules with a high activity of functionallysubstituting for F.VIII and a low F.Xase inhibitory action, which areantibodies that recognize both an enzyme and its substrate.

Since humanized antibodies may generally have high stability in bloodand low immunogenicity, multispecific antibodies of the presentinvention may be very promising as pharmaceuticals.

1-29. (canceled)
 30. A multi-specific antigen-binding molecule thatfunctionally substitutes for blood coagulation factor VIII and comprises(a) an antigen-binding site that recognizes blood coagulation factor IXand/or activated blood coagulation factor IX, and (b) an antigen-bindingsite that recognizes blood coagulation factor X, wherein themulti-specific antigen-binding molecule is more active in promotinggeneration of activated blood coagulation factor X than is a bispecificantibody that comprises an antibody H chain comprising SEQ ID NO: 165,an antibody H chain comprising SEQ ID NO: 166, and two antibody Lchains, each comprising SEQ ID NO:
 167. 31. The multi-specificantigen-binding molecule of claim 30, which comprises: a firstpolypeptide comprising an antigen-binding site that recognizes bloodcoagulation factor IX and/or activated blood coagulation factor IX; asecond polypeptide comprising an antigen-binding site that recognizesblood coagulation factor X; a third polypeptide comprising anantigen-binding site that recognizes blood coagulation factor IX and/oractivated blood coagulation factor IX; and a fourth polypeptidecomprising an antigen-binding site that recognizes blood coagulationfactor X.
 32. The multi-specific antigen-binding molecule of claim 31,wherein: the first polypeptide comprises an antigen-binding site of afirst antibody H chain; the second polypeptide comprises anantigen-binding site of a second antibody H chain; the third polypeptidecomprises an antigen-binding site of a first antibody L chain; and thefourth polypeptide comprises an antigen-binding site of a secondantibody L chain, wherein the first antibody H chain and first antibodyL chain can associate to form an antibody that binds to bloodcoagulation factor IX or activated blood coagulation factor IX, whereinthe second antibody H chain and the second antibody L chain canassociate to form an antibody that binds to blood coagulation factor X,and wherein the first and second antibody L chains can be the same ordifferent.
 33. The multi-specific antigen-binding molecule of claim 32,wherein: the first antibody H chain comprises a set of antibody H chainCDRs 1, 2, and 3 selected from the following sets of CDRs 1, 2 and 3:SEQ ID NOs: 75-77, respectively, SEQ ID NOs: 78-80, respectively, SEQ IDNOs: 81-83, respectively, SEQ ID NOs: 84-86, respectively, SEQ ID NOs:87-89, respectively, SEQ ID NOs: 90-92, respectively, SEQ ID NOs: 93-95,respectively, SEQ ID NOs: 96-98, respectively, SEQ ID NOs: 99-101,respectively, SEQ ID NOs: 102-104, respectively, and SEQ ID NOs:105-107, respectively; and (2) the second polypeptide comprises a set ofantibody H chain CDRs 1, 2, and 3 selected from the following sets ofCDRs 1, 2 and 3: SEQ ID NOs: 108-110, respectively, SEQ ID NOs: 111-113,respectively, SEQ ID NOs: 114-116, respectively, SEQ ID NOs: 117-119,respectively, SEQ ID NOs: 120-122, respectively, SEQ ID NOs: 123-125,respectively, SEQ ID NOs: 126-128, respectively, SEQ ID NOs: 129-131,respectively, SEQ ID NOs: 132-134, respectively, SEQ ID NOs: 135-137,respectively, and SEQ ID NOs: 174-176, respectively.
 34. Themulti-specific antigen-binding molecule of claim 32, wherein: the firstpolypeptide comprises an antibody H chain variable region comprising anyone of SEQ ID NOs: 35-45; and the second polypeptide comprises anantibody H chain variable region comprising any one of SEQ ID NOs: 46,47, 48, 49, 50, 51, 52, 53, 54, 55, and
 172. 35. The multi-specificantigen-binding molecule of claim 32, wherein each of the third andfourth polypeptides comprises a set of antibody L chain CDRs 1, 2, and 3independently selected from the following sets of CDRs 1, 2, and 3: SEQID NOs: 138-140, respectively, SEQ ID NOs: 141-143, respectively, SEQ IDNOs: 144-146, respectively, SEQ ID NOs: 147-149, respectively, SEQ IDNOs: 150-152, respectively, SEQ ID NOs: 153-155, respectively, SEQ IDNOs: 156-158, respectively, SEQ ID NOs: 159-161, respectively, SEQ IDNOs: 137-139, respectively, and SEQ ID NOs: 177-179, respectively. 36.The multi-specific antigen-binding molecule of claim 32, wherein each ofthe third and fourth polypeptides comprises an antibody L chain variableregion comprising a sequence independently selected from SEQ ID NOs: 56,57, 58, 59, 60, 61, 62, 63, 64, and
 173. 37. The multi-specificantigen-binding molecule of claim 32, wherein each of the first andsecond polypeptides comprises an antibody H chain constant region thatmay be the same or different, and each of the third and fourthpolypeptides comprises an antibody L chain constant region that may bethe same or different.
 38. The multi-specific antigen-binding moleculeof claim 37, wherein the third and fourth polypeptides are identicalantibody L chains.
 39. The multi-specific antigen-binding molecule ofclaim 37, wherein: the first polypeptide comprises an antibody H chainconstant region of any one of SEQ ID NOs: 65-73; and the secondpolypeptide comprises an antibody H chain constant region of (i) any oneof SEQ ID NOs: 65-70 when the first polypeptide comprises an antibody Hchain constant region of one of SEQ ID NOs: 71-73, or (ii) any one ofSEQ ID NOs: 71-73 when the first polypeptide comprises an antibody Hchain constant region of one of SEQ ID NOs: 65-70.
 40. Themulti-specific antigen-binding molecule of claim 37, wherein each of thethird and fourth polypeptides comprises an antibody L chain constantregion consisting of SEQ ID NO:
 74. 41. The multi-specificantigen-binding molecule of claim 37, wherein: the first polypeptidecomprises an antibody H chain selected from any one of SEQ ID NOs: 1-3and 10-20; the second polypeptide comprises an antibody H chain selectedfrom any one of SEQ ID NOs: 4-7, 21-27, and 170; and each of the thirdand fourth polypeptides comprises an antibody L chain independentlyselected from SEQ ID NOs: 8, 9, 28-34, and 171, wherein the third andfourth polypeptides may be the same or different.
 42. The multi-specificantigen-binding molecule of claim 30, wherein: the first polypeptidebinds to an epitope overlapping with an epitope bound by an antibodyconsisting of (i) an antibody H chain consisting of any one of SEQ IDNOs: 1-3 and 10-20, and (ii) an antibody L chain consisting of any oneof SEQ ID NOs: 8, 9, 28-34, and 171; and the second polypeptide binds toan epitope overlapping with an epitope bound by an antibody consistingof (iii) an antibody H chain consisting of any one of SEQ ID NOS: 4-7,21-27, and 170, and (iv) an antibody L chain consisting of any one ofSEQ ID NOs: 8, 9, 28-34, and
 171. 43. The multi-specific antigen-bindingmolecule of claim 37, wherein: the first polypeptide is: (a) an H chainof an antibody that binds to an epitope overlapping with an epitopebound by an antibody that consists of (A) an antibody H chain consistingof any one of SEQ ID NOs: 1-3 and 10-20, and (B) an antibody L chainconsisting of any one of SEQ ID NOs: 8, 9, 28-34, and 171; or (b) anantibody H chain that corresponds to any one of (a), except that atleast one amino acid residue selected from the amino acid residues atpositions 34, 35, 49, 61, 62, 96, 98, 100, 100b, and 102 (by Kabatnumbering) is substituted with another amino acid residue; the secondpolypeptide is: (c) an H chain of an antibody that binds to an epitopeoverlapping with an epitope bound by an antibody that consists of (A) anantibody H chain consisting of any one of SEQ ID NOs: 4-7, 21-27, and170, and (B) an antibody L chain consisting of any one of SEQ ID NOs: 8,9, 28-34, and 171; or (d) an antibody H chain that corresponds to anyone of (c), except that at least one amino acid residue selected fromthe amino acid residues at positions 35, 53, 73, 76, 96, 98, 100, and100a (by Kabat numbering) is substituted with another amino acidresidue; and the third and fourth polypeptides are independentlyselected from any of the antibody L chains of (e), (f), and (g): (e) anL chain of an antibody that binds to an epitope overlapping with anepitope bound by an antibody that consists of (A) an antibody H chainconsisting of any one of SEQ ID NOs: 1-3 and 10-20, and (B) an antibodyL chain consisting of any one of SEQ ID NOs: 8, 9, 28-34, and 171; (f)an L chain of an antibody that binds to an epitope overlapping with anepitope bound by an antibody that consists of (A) an antibody H chainconsisting of any one of SEQ ID NOs: 4-7, 21-27, and 170, and (B) anantibody L chain consisting of any one of SEQ ID NOs: 8, 9, 28-34, and171; and (g) an antibody L chain that corresponds to any one of (e) and(f), except that at least one amino acid residue selected from the aminoacid residues at positions 27, 30, 31, 32, 50, 52, 53, 54, 55, 92, 93,94, and 95 (by Kabat numbering) is substituted with another amino acid.44. The multi-specific antigen-binding molecule of claim 43, wherein:the first polypeptide is an antibody H chain that corresponds to (a) or(b) of claim 43 and comprises any one or more of an isoleucine at aminoacid position 34; an asparagine, glutamine, or serine at amino acidposition 35; a serine at amino acid position 49; an arginine at aminoacid position 61; a glutamic acid at amino acid position 62; a serine orthreonine at amino acid position 96; a lysine or arginine at amino acidposition 98; a phenylalanine or tyrosine at amino acid position 100; aglycine at amino acid position 100b; and a tyrosine at amino acidposition 102 (all positions by Kabat numbering); the second polypeptideis an antibody H chain that corresponds to (c) or (d) of claim 43 andcomprises any one or more of an aspartic acid at amino acid position 35;an arginine at amino acid position 53; a lysine at amino acid position73; a glycine at amino acid position 76; a lysine or arginine at aminoacid position 96; a tyrosine at amino acid position 98; a tyrosine atamino acid position 100; and a histidine at amino acid position 100a(all positions by Kabat numbering); and/or the third and fourthpolypeptides are antibody L chains, each independently corresponding toone of (e), (f), and (g) of claim 43 and comprising any one of more of alysine or arginine at amino acid position 27; a glutamic acid at aminoacid position 30; an arginine at amino acid position 31; a glutamine atamino acid position 32; an arginine or glutamine at amino acid position50; a serine at amino acid position 52; an arginine at amino acidposition 53; a lysine at amino acid position 54; a glutamic acid atamino acid position 55; a serine at amino acid position 92; a serine atamino acid position 93; a proline at amino acid position 94; and aproline at amino acid position 95 (all positions by Kabat numbering).45. The multi-specific antigen-binding molecule of claim 30, wherein themulti-specific antigen-binding molecule is a multi-specific antibody.46. A bispecific antibody comprising a first polypeptide, a secondpolypeptide, a third polypeptide, and a fourth polypeptide, wherein: (a)the first polypeptide is an antibody H chain consisting of SEQ ID NO: 1,the second polypeptide is an antibody H chain consisting of SEQ ID NO:4, and the third and fourth polypeptides are antibody L chains, eachconsisting of SEQ ID NO: 9; (b) the first polypeptide is an antibody Hchain consisting of SEQ ID NO: 1, the second polypeptide is an antibodyH chain consisting of SEQ ID NO: 5, and the third and fourthpolypeptides are antibody L chains, each consisting of SEQ ID NO: 9; (c)the first polypeptide is an antibody H chain consisting of SEQ ID NO: 2,the second polypeptide is an antibody H chain consisting of SEQ ID NO:6, and the third and fourth polypeptides are antibody L chains, eachconsisting of SEQ ID NO: 8; (d) the first polypeptide is an antibody Hchain consisting of SEQ ID NO: 3, the second polypeptide is an antibodyH chain consisting of SEQ ID NO: 7, and the third and fourthpolypeptides are antibody L chains, each consisting of SEQ ID NO: 9; (e)the first polypeptide is an antibody H chain consisting of SEQ ID NO:10, the second polypeptide is an antibody H chain consisting of SEQ IDNO: 6, and the third and fourth polypeptides are antibody L chains, eachconsisting of SEQ ID NO: 30; (f) the first polypeptide is an antibody Hchain consisting of SEQ ID NO: 10, the second polypeptide is an antibodyH chain consisting of SEQ ID NO: 7, and the third and fourthpolypeptides are antibody L chains, each consisting of SEQ ID NO: 33;(g) the first polypeptide is an antibody H chain consisting of SEQ IDNO: 11, the second polypeptide is an antibody H chain consisting of SEQID NO: 4, and the third and fourth polypeptides are antibody L chains,each consisting of SEQ ID NO: 33; (h) the first polypeptide is anantibody H chain consisting of SEQ ID NO: 11, the second polypeptide isan antibody H chain consisting of SEQ ID NO: 5, and the third and fourthpolypeptides are antibody L chains, each consisting of SEQ ID NO: 30;(i) the first polypeptide is an antibody H chain consisting of SEQ IDNO: 12, the second polypeptide is an antibody H chain consisting of SEQID NO: 21, and the third and fourth polypeptides are antibody L chains,each consisting of SEQ ID NO: 33; (j) the first polypeptide is anantibody H chain consisting of SEQ ID NO: 13, the second polypeptide isan antibody H chain consisting of SEQ ID NO: 22, and the third andfourth polypeptides are antibody L chains, each consisting of SEQ ID NO:29; (k) the first polypeptide is an antibody H chain consisting of SEQID NO: 14, the second polypeptide is an antibody H chain consisting ofSEQ ID NO: 21, and the third and fourth polypeptides are antibody Lchains, each consisting of SEQ ID NO: 33; (l) the first polypeptide isan antibody H chain consisting of SEQ ID NO: 15, the second polypeptideis an antibody H chain consisting of SEQ ID NO: 23, and the third andfourth polypeptides are antibody L chains, each consisting of SEQ ID NO:30; (m) the first polypeptide is an antibody H chain consisting of SEQID NO: 16, the second polypeptide is an antibody H chain consisting ofSEQ ID NO: 21, and the third and fourth polypeptides are antibody Lchains, each consisting of SEQ ID NO: 28; (n) the first polypeptide isan antibody H chain consisting of SEQ ID NO: 17, the second polypeptideis an antibody H chain consisting of SEQ ID NO: 27, and the third andfourth polypeptides are antibody L chains, each consisting of SEQ ID NO:34; (o) the first polypeptide is an antibody H chain consisting of SEQID NO: 18, the second polypeptide is an antibody H chain consisting ofSEQ ID NO: 25, and the third and fourth polypeptides are antibody Lchains, each consisting of SEQ ID NO: 30; (p) the first polypeptide isan antibody H chain consisting of SEQ ID NO: 19, the second polypeptideis an antibody H chain consisting of SEQ ID NO: 24, and the third andfourth polypeptides are antibody L chains, each consisting of SEQ ID NO:30; (q) the first polypeptide is an antibody H chain consisting of SEQID NO: 19, the second polypeptide is an antibody H chain consisting ofSEQ ID NO: 24, and the third and fourth polypeptides are antibody Lchains, each consisting of SEQ ID NO: 31; (r) the first polypeptide isan antibody H chain consisting of SEQ ID NO: 19, the second polypeptideis an antibody H chain consisting of SEQ ID NO: 27, and the third andfourth polypeptides are antibody L chains, each consisting of SEQ ID NO:28; (s) the first polypeptide is an antibody H chain consisting of SEQID NO: 20, the second polypeptide is an antibody H chain consisting ofSEQ ID NO: 25, and the third and fourth polypeptides are antibody Lchains, each consisting of SEQ ID NO: 32; (t) the first polypeptide isan antibody H chain consisting of SEQ ID NO: 20, the second polypeptideis an antibody H chain consisting of SEQ ID NO: 26, and the third andfourth polypeptides are antibody L chains, each consisting of SEQ ID NO:31; and (u) the first polypeptide is an antibody H chain consisting ofSEQ ID NO: 12, the second polypeptide is an antibody H chain consistingof SEQ ID NO: 170, and the third and fourth polypeptides are antibody Lchains, each consisting of SEQ ID NO:
 171. 47. A nucleic acid encodingthe multi-specific antigen-binding molecule of claim
 30. 48. A vectorcomprising the nucleic acid of claim
 47. 49. A cell comprising thenucleic acid of claim
 47. 50. A method for producing the multi-specificantigen-binding molecule by culturing the cell of claim
 49. 51. Apharmaceutical composition comprising the multi-specific antigen-bindingmolecule of claim 30 and a pharmaceutically acceptable carrier.
 52. Amethod for treating or reducing the incidence of bleeding, a diseaseaccompanying bleeding, or a disease caused by bleeding in a subject, themethod comprising identifying a subject as being in need of thereof andadministering a therapeutically effective amount of the pharmaceuticalcomposition of claim 51 to the subject.
 53. The method of claim 52,wherein the bleeding, the disease accompanying bleeding, or the diseasecaused by bleeding is a disease that develops and/or progresses due to adecrease or deficiency in the activity of blood coagulation factor VIIIand/or activated blood coagulation factor VIII.
 54. The method of claim53, wherein the disease that develops and/or progresses due to adecrease or deficiency in the activity of blood coagulation factor VIIIand/or activated blood coagulation factor VIII is hemophilia A.
 55. Themethod of claim 53, wherein the disease that develops and/or progressesdue to a decrease or deficiency in the activity of blood coagulationfactor VIII and/or activated blood coagulation factor VIII is a diseaseshowing emergence of an inhibitor against blood coagulation factor VIIIand/or activated blood coagulation factor VIII.
 56. The method of claim53, wherein the disease that develops and/or progresses due to adecrease or deficiency in the activity of blood coagulation factor VIIIand/or activated blood coagulation factor VIII is acquired hemophilia.57. The method of claim 53, wherein the disease that develops and/orprogresses due to a decrease in the activity of blood coagulation factorVIII and/or activated blood factor VIII is von Willebrand disease.
 58. Akit comprising the multi-specific antigen-binding molecule of claim 30.